Model for neurodegenerative diseases involving amyloid accumulation

ABSTRACT

The present invention provides brain cells, such as normal brain cells, apolipoprotein E deficient brain cells, or apoE4 containing brain cells, that are treated with a compound which can modulate integrins and/or integrin receptors to produce increased sequestration of and/or accumulation of and/or uptake of Aβ, and/or changes in cathepsin D content and/or lysosomal dysfunction, and/or microglia activation in the brain cells. The present invention also provides methods for producing such cells and methods for using the cells for screening an agent or substance that modulates the sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or changes in cathepsin D content and/or microglia activation in the brain cells. The method further provides a new therapeutic target, antagonism of glutamate receptors, for the treatment of neurodegenerative diseases which are characterized by inter alia, abnormal amyloid uptake and/or accumulation.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/235,374 filed Sep. 25, 2000, the contents of which is incorporatedherein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No.455365-30110, awarded by the National Institute of Aging. The Governmentmay have certain rights in this invention

FIELD OF THE INVENTION

The invention is in the field of models and interventions of medicaldiseases. Specifically, the invention is in the field ofneurodegenerative disease models and treatments, and especially agerelated neurodegenerative diseases such as Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a leading cause of dementia in the elderly,affecting 5-10% of the population over the age of 65 years. See A Guideto Understanding Alzheimer's Disease and Related Disorders, edited byJorm, New York University Press, New York (1987). AD currently affects12 million people around the world, and is projected to affect 22million people by 2025 and 45 million by 2050.

Alzheimer's disease is histopathologically characterized by the loss ofparticular groups of neurons and the appearance of two principal lesionswithin the brain, termed senile plaques and neurofibrillary tangles. SeeBrion et al., J. Neurochem. 60:1372-1382 (1993). Neurofibrillary tanglesare intraneuronal accumulations of an abnormally phosphorylated form ofthe microtubule protein tau. Neurofibrillary tangles are most abundantlypresent in parts of the brain associated with memory functions, such asthe hippocampus and adjacent parts of the temporal lobe. See RobbinsPathologic Basis of Disease, Cotran et al., 6th ed., W.B. SaundersCompany (1999), p. 1300.

Amyloid beta peptides (Aβ) are normally secreted proteolytic products ofamyloid precursor protein (APP) (Selkoe, D. J., Annu Rev Cell Biol10:373-403 (1994)). The 42-residue form (Aβ1-42) is the principalspecies in senile plaques which constitute a diagnostic feature ofAlzheimer's disease (AD), and is preferentially generated over shorterforms (e.g., Aβ1-40) in genetic mutations related to familial AD(Steiner, H., et al., Eur Arch Psychiatry Clin Neurosci 249:266-270(1999)). Transgenic mice that overexpress mutant APP develop plaquesaccompanied by neuropathology (Guenette, S. Y., and Tanzi, R. E.,Neurobiol Aging 20:201-11 (1999); van Leuven, 2000) with both effectsbeing blocked by immunization against Aβ (Schenk, D., et al., Nature400:173-177 (1999); Frenkel, D., et al., Proc Natl Acad Sci USA97:11455-11459 (2000); Janus, C., et al., Biochim Biophys Acta1502:63-75 (2000); Morgan, D., et al., Nature 408:982-85 (2000)), or byperipheral administration of antibodies against Aβ (Bard, F., et al.,Nat Med 6:916-9 (2000)).

These results support the assumption that extracellular Aβ accumulationstrigger pathologies and emphasize the importance of identifying linksbetween the peptide and pathogenic processes. Infusions of Aβ into braindo not cause extensive damage (Games, D., et al., Neurobiol Aging13:569-576 (1992); Podlisny, M. B., et al., Am J Pathol 142:17-24(1993)), in part, because extracellular proteases prevent the injectedmaterial from assembling into plaques (Backstrom, J. R., et al., JNeurosci 16:7910-7919 (1996); Qiu, W. Q., et al., J Biol Chem273:32730-32738 (1998); Caswell, M. D., et al., Eur J Biochem266:509-516 (1999); Iwata, N., et al., Nature Med 6:143-150 (2000);Vekrellis, K., et al., J Neurosci 20:1657-1665 (2000)). In any event,links between extracellular Aβ and pathogenic mechanisms in mature brainremain obscure.

Brain slices in interface culture reach a surprisingly adult-like state(Stoppini, L., et al., J Neurosci Meth 37:173-182 (1991); Muller, D., etal., Dev Brain Res 71:93-100 (1993); Bahr, B. A., et al., Hippocampus5:425-439 (1995)) and offer opportunities for in vitro studies of brainaging. Initial studies using this model found that Aβ treatmentmoderately enhanced cell death (Bruce, A., et al., Proc Nat Acad Sci93:2312-2316 (1996)), while a later study found little effect of Aβ1-42on measures of pathogenesis (Bahr, B., et al., J Comp Neurol 397:139-147(1998)). A third study confirmed that Aβalone did not cause pathologybut in combination with transforming growth factor-β induced neuronaldegeneration in field CA1 (Harris-White, M. E., et al., J Neurosci18:10366-10374 (1998)).

The relatively weak effects of Aβ on cultured slices could reflect slowinternalization and modest accumulation. Uptake of Aβ1-42 in culturedhippocampal slices occurs selectively in field CA1 (Bahr, B., et al., JComp Neurol 397:139-147 (1998); Harris-White, M. E., et al., J Neurosci18:10366-10374 (1998)), suggesting the existence of regionallydifferentiated factors that govern sequestration and regulate toxicityof the peptide. Integrins mediate internalization of bacteria andviruses (Isberg, R. R., and Tran Van Nhieu, G., Trends Microbiol 2:10-14(1994); Nemerow, G. R., and Stewart, P. L., Microbiol Mol Biol Rev63:725-734 (1999)) and bind Aβ via an Arg-Gly-Asp (RGD)-like sequence(Ghiso, J., et al., Biochem J 288:1053-1059 (1992); Sabo, S., et al.,Neurosci Lett 184:25-28 (1995); Yamazaki, T., et al., J Neurosci17:1004-1010 (1997)). Binding to alpha5 β1 integrin, a fibronectinreceptor densely expressed in hippocampus (Bahr, B., et al., Neuroreport2:13-16 (1991); Bahr, B. and Lynch, G., Biochem J 281:137-142 (1992);Pinkstaff, J. K., et al., J Neurosci 19:1541-1556 (1999); Bi, X., etal., J Comp Neurol 435:184-193 (2001)) is required for Aβinternalization in cell lines (Matter, M. L., et al., J Cell Biol141:1019-1030 (1998)). Moreover, the different subdivisions ofhippocampus express different combinations of integrins (Pinkstaff, J.K., et al., J Neurosci 19:1541-1556 (1999)), an anatomical feature thatcould account for regional variations in Aβ uptake.

Integrins interact with neighboring transmembrane proteins to producetheir effects on cell surface operations (Burkin, D. J., et al., J CellBiol 143:1067-1075 (1998); Porter, J. C., and Hogg, N., Trends CellBiol. 8:390-396 (1998)) including calcium influx (Tsao, P. W., andMousa, S. A., J Biol Chem 270:23742-23753 (1995); Wu, X., et al., J CellBiol 143:241-252 (1998)). NMDA receptors are calcium permeant, coupledto the actin cytoskeleton (Dunah, A. W., et al., Brain Res Mol Brain Res79:77-87 (2000)); both calcium (Marsh, M., and McMahon, H. T., Science285:215-220 (1999)) and the actin network (Gottlieb, T. A., et al., JCell Biol 120:695-710 (1993)) are crucial to endocytosis. NMDA receptorfunction (Bahr, B. A., J Neurosci Res 59:827-832 (2000)) and maturationof synapses containing NMDA receptors (Chavis, P., and Westbrook, G.,Nature 411:317-321 (2001)) have been shown to be modulated by integrins.Finally, recent studies indicate that NMDA receptor activation cantrigger clathrin-mediated internalization (Carroll, R. C., Proc NatlAcad Sci USA 96:14112-14117 (1999); Beattie, E. C., et al., Nat Neurosci3:1291-300 (2000); Ehlers, M. D., Neuron 28:511-25 (2000)).

There has been considerable research into mechanisms underlyingneurodegenerative diseases, including Alzheimer's disease. For example,many transgenic animal models of Alzheimer's disease have been developedand used in an attempt to study the mechanisms of Alzheimer's disease aswell as to screen compounds that may ameliorate the conditions ofAlzheimer's disease. However, many in vivo or in vitro models are unableto produce some of the important features of Alzheimer's disease, suchas neurofibrillary tangles, microglia activation, lysosomal dysfunction,intracellular and/or extracellular sequestration and/or uptake and/oraccumulations of amyloid, etc. Thus, there is an ongoing need to developa model that better mimics the pathologies associated withneurodegenerative diseases including Alzheimer's disease and new ways toinvestigate and combat such conditions.

The present invention provides a model that better mimics some of thepathologies of neurodegenerative diseases, including Alzheimer'sdisease, than other models known in the art. The present invention meetsthese and other needs, and also provides new ways to investigate andcombat such neurodegenerative conditions. Related to the presentinvention is U.S. application Ser. No. 09/917,789 which is incorporatedby reference herein in its entirety

SUMMARY OF THE INVENTION

The present invention provides a model for neurodegenerative diseases,including Alzheimer's disease and other age-related neuro-degenerativediseases, wherein the model provides brain cells, or brain tissuecontaining the same. The invention further provides a method forincreasing or decreasing characteristics and changes indicative ofneurodegenerative diseases in such cells. These changes especiallyinclude increasing sequestration of and/or accumulation of and/or uptakeof Aβ, and/or lysosomal dysfunction, and/or microglia activation. Thepresent invention also provides a model wherein brain cells comprise amarked microglia activation and increases in the levels and/or activityof cathepsin D. As described above, many currently available in vivo andin vitro models of neurodegenerative diseases and aged brain lack someor all of these key features.

The present invention is based on, in part, the discovery that integrinsand/or integrin receptors can modulate the sequestration and/oraccumulation and/or uptake of Aβ in cultured brain cells. Further, thepresent invention is also based upon the discovery that glutamatereceptors within the brain, for example the NMDA-subtype of glutamatereceptors, can modulate the sequestration and/or accumulation and/oruptake of Aβ in cultured brain cells. Specifically, the treatment ofbrain cells with agent(s) capable of modulating integrins and/orintegrin receptors surprisingly triggered the sequestration and/oraccumulation and/or uptake of Aβ. Further, agent(s) which affectglutamate receptors within the brain, for example the NMDA-subtype ofglutamate receptors, blocked the modulation of the sequestration and/oraccumulation and/or uptake of Aβ in brain cells treated with agent(s)capable of modulating integrins and/or integrin receptors. These resultscan be observed in any suitable brain cells including, e.g., normalbrain cells, brain cells derived from transgenic animals, etc.

Among many types of brain cells suitable for embodiments of theinvention, hippocampal brain cells treated with agent(s) capable ofmodulating integrins and/or integrin receptors produced sequestrationand/or accumulations and/or uptake of Aβ at significantly enhancedlevels when compared with hippocampal brain cells not treated withagent(s) capable of modulating integrins and/or integrin receptors. Theenhanced sequestration and/or accumulations and/or uptake of Aβ aretypically formed within a few days of treatment, and morphologicallymimic early stage amyloid sequestration and/or accumulations and/oruptake found in the brains of Alzheimer's patients. Such levels ofsequestration and/or accumulations and/or uptake of Aβ was notachievable in brain cells in vitro even with prolonged treatment with Aβwithout the presence of agent(s) capable of modulating integrins and/orintegrin receptors. Therefore, if brain cells with robust sequestrationand/or accumulations and/or uptake of Aβ is desired, the use of agent(s)capable of modulating integrins and/or integrin receptors can bepreferably used in embodiments of the invention. Thus, the presentinvention provides, among other things, brain cells in vitro comprisingenhanced levels of sequestration and/or accumulations and/or uptake ofAβ which can be used as a model for neurodegenerative diseases,including Alzheimer's disease.

Accordingly, in one aspect, the invention provides an in vitro method ofincreasing sequestration and/or accumulation and/or uptake of Aβ, themethod comprising: (a) contacting cultured brain cells with agent(s)capable of modulating integrins and/or integrin receptors; and (b)determining the sequestration of and/or accumulation of and/or uptake ofAβ in the cell culture.

In another aspect, the invention provides a method comprising,contacting brain cells with a compound that is capable of modulatingintegrins and/or integrin receptors, thereby producing properties of abrain afflicted with a neurodegenerative disease, wherein the propertiesinclude increased sequestration of and/or accumulation of and/or uptakeof Aβ.

In yet another aspect, the invention provides brain cells in vitro thathave been cultured in a medium capable of modulating integrins and/orintegrin receptors in the brain cells, wherein the brain cells comprisean increased amount of sequestration of and/or accumulation of and/oruptake of Aβ compared to a control.

In yet another aspect, the invention provides brain cells in vitro,wherein the brain cells have been treated with a compound that iscapable of modulating integrins and/or integrin receptors, therebyproducing properties of a brain afflicted with a neurodegenerativedisease, wherein the properties include increased sequestration ofand/or accumulation of and/or uptake of Aβ.

In yet another aspect, the invention provides a screening methodcomprising: (a) contacting brain cells in vitro, with a compound that iscapable of modulating integrins and/or integrin receptors in the braincells, wherein the compound is capable of increasing the sequestrationof and/or accumulation of and/or uptake of Aβ; (b) contacting the braincells with an agent; and (c) determining whether the agent modulates theamount of sequestration of and/or accumulation of and/or uptake of Aβ inthe brain cells treated with the agent compared to the brain cells thatare not treated with the agent.

In yet another aspect, the invention provides a method of increasing thesequestration of and/or accumulation of and/or uptake of Aβ in anysuitable brain cells, the method comprising: (a) contacting the braincells in a medium which is capable of modulating integrins and/orintegrin receptors; and (b) determining the sequestration of and/oraccumulation of and/or uptake of Aβ in the brain cells.

In another aspect, the invention provides a method comprising: (a)culturing brain cells; and (b) contacting the brain cells with acompound which is capable of modulating integrins and/or integrinreceptors, thereby producing properties of a brain afflicted with aneurodegenerative disease, wherein the properties include increasedsequestration of and/or accumulation of and/or uptake of Aβ.

In yet another aspect, the invention provides brain cells in vitro thathave been cultured in a medium which modulates integrins and/or integrinreceptors in the brain cells, wherein the brain cells comprise anincreased sequestration of and/or accumulation of and/or uptake of Aβcompared to a control.

In yet another aspect, the invention provides brain cells in vitro,wherein the brain cells have been treated with a compound that modulatesintegrins and/or integrin receptors in the brain cells, therebyproducing properties of a brain afflicted with a neurodegenerativedisease, wherein the properties include increased sequestration ofand/or accumulation of and/or uptake of Aβ.

In yet another aspect, the invention provides a screening methodcomprising: (a) contacting brain cells in vitro, with a compound thatmodulates integrins and/or integrin receptors in the brain cells,wherein the modulation of integrins and/or integrin receptors is capableof increasing the sequestration of and/or accumulation of and/or uptakeof Aβ in the brain cells; (b) contacting the brain cells with an agent;and (c) determining whether the agent modulates the amount ofsequestration of and/or accumulation of and/or uptake of Aβ in the braincells treated with the agent compared to the brain cells that are nottreated with the agent.

In yet another aspect, the invention provides a method for determiningthe effect of a substance on characteristics of neurodegenerativedisease in brain cells, said method comprising: (A) exposing brain cellsto a condition that modulates integrins or integrin receptors in saidcells, (B) maintaining said cells for a time sufficient to induce one ormore characteristics of a neurodegenerative disease in said cells, (C)adding said substance before, during and/or after said exposing ormaintaining; and (D) determining whether the presence of said substancehas an effect on one or more of said characteristics.

Another aspect of the invention provides method of obtaining brain cellshaving characteristics of neurodegenerative disease comprising (A)culturing brain cells, (B) exposing said brain cells to a condition thatmodulates integrins or integrin receptors in said cells, and (C)maintaining said cells or brain tissue for a time sufficient to induceone or more characteristics of a neurodegenerative disease in saidcells.

Another aspect of the invention is directed to an in vitro method forincreasing at least one or more characteristics of neurodegenerativedisease in brain cells, wherein said characteristics are selected fromthe group consisting sequestration of Aβ, accumulation of Aβ, uptake ofAβ, lysosomal dysfunction and microglia activation, said in vitro methodcomprising: (A) exposing brain cells in culture to a condition thatmodulates integrins or integrin receptors in said cells wherein saidmodulation results in increase in characteristics of neurodegenerativedisease in said cells, and (B) maintaining said cells in culture for atime sufficient to increase one or more characteristics of aneurodegenerative disease in said cells.

Another aspect of the invention is directed to a method for determiningthe effect of a substance on inhibition of characteristics ofneurodegenerative disease in brain cells, said method comprising: (A)exposing brain cells to a condition that modulates integrins or integrinreceptors in said cells, (B) maintaining said cells for a timesufficient to induce one or more characteristics of a neurodegenerativedisease in said cells, (C) adding said substance before, during and/orafter said exposing or maintaining; and (D) determining whether thepresence of said substance inhibits one or more of said characteristics.

The invention is also directed to a method for determining the effect ofa substance on inhibition of characteristics of neurodegenerativedisease in brain cells, said method comprising: (A) exposing brain cellsto a condition that modulates integrins or integrin receptors in saidcells, (B) maintaining said cells for a time sufficient to induce one ormore characteristics of a neurodegenerative disease in said cells, (C)adding said substance before, during and/or after said exposing ormaintaining; and (D) determining whether the presence of said substanceinhibits one or more of said characteristics.

Aspects of the invention include methods drawn to the effect of thesubstance on characteristics selected from the group consisting ofsequestration of Aβ, accumulation of Aβ, uptake of Aβ and lysosomaldysfunction, changes in cathepsin D content and microglia activation.Additional embodiments look at increases or decreases of thesecharacteristics, such as where the changes are at least about 10%compared to a control.

The methods of the invention are also drawn to obtaining brain cells andthe use of brains cells wherein the brain cells may be in vivo or invitro such as in the form of a brain slice. The brain slice may includea hippocampal slice, an entorhinal cortex slice, an entorhinohippocampalslice, a neocortex slice, a hypothalamic slice, or a cortex slice. Braincells may also be obtained from a non-human transgenic animal. Suchanimals may comprise a human apolipoprotein E4 gene or and animal wherean endogenous apolipoprotein E gene of the non-human transgenic animalare ablated. Apolipoprotein E deficient brain cells or apolipoprotein E4containing brain cells cultured in a medium which selectively increasesare also included in the invention.

The methods of the invention are also drawn to culturing brain slices orthe cells therein in a medium that comprises an antagonist or modulatorof an integrin. The modulator or antagonist may be selected from thegroup consisting of neutralizing and/or function blocking antibodies forintegrin subunits alpha1, alpha2, alpha3, alpha4, alpha5, alpha6,alpha7, alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 andbeta8. The methods of the invention are further drawn to a peptideselected from the group of peptides consisting of RGD, RGDS (SEQ. IDNO.1), GRGDS (SEQ. ID NO.2), GRGDSP (SEQ. ID NO.3), GRGDTP (SEQ. IDNO.4), mimetics thereof and disintegrins such as echistatin found insnake venom.

The methods of the invention are also directed to determining visuallythe amount of: sequestration of Aβ, accumulation of Aβ, uptake of Aβ,lysosomal dysfunction or microglia activation is determined visually.The determinations may be done visually and also using a capturereagent. The capture reagents may include is an antibody that binds toAβ, lysosomes, cathepsin D or a microglia element.

The methods of the invention are also directed to either contacting thebrain cells simultaneously with the compound that modulates integrinsand/or integrin receptors or contacting the brains cells with thecompound that modulates integrins and/or integrin receptors prior tocontact with the substance whose effect is being determined.

Another aspect of the invention is directed to a method for alleviatingthe symptoms of disease states having at least one of the followingcharacteristics selected from the group consisting of intracellularuptake of amyloid protein, amyloid accumulation and/or plaque formation,said method comprising: (A) administering to a patient in need thereof acomposition comprising an effective amount of an NMDA receptorantagonist, and (B) determining the effectiveness of treatment with saidcomposition, (C) increasing or decreasing the composition based on thedeterminative testing, and (D) alleviating symptoms of the disease.Determinative testing may include methods of brain imaging such as MRIor PET. Additional, determinative testing may includeelectroencephalogram analysis as well as cognitive testing.

The invention is also directed to a pharmaceutical compositioncomprising a compound capable of sufficiently inhibiting the activity ofthe NMDA receptors in an amount effective to alleviate one or moresymptoms of disease states associated with at least one characteristicselected from the group consisting of abnormal accumulation, abnormalmolecular organization of amyloid protein and/or amyloid plaques andsaid composition also includes a suitable carrier or pharmaceuticalexcipient. Embodiments of the invention may include a compositioncomprising at least one of the compounds selected from a groupconsisting of magnesium, ketamine, dextromethorphan, amantadine,dexanabinol, AP3, AP5, AP6, AP7, 4C3HPG, 4CPG, CGS 19755,chlorophenylglutamic acid, CPP, MK-801, PCP, ibogaine, noribogaine,ifenprodil, flupirtine, selfotel, D-CPP-ene, procyclidine,trihexyphenidyl, CP-101606, CP-98113, GV1150526, AR-R15896AR, NPS 1506,NPC 12626, LY274614, LY 2835959, SDZ 220-040, SDZ 220-040, SDZ 220-581,SDZ 221-653 and memantine.

Another aspect of the invention is directed to a method for inhibitingthe intracellular accumulation of amyloid comprising: (A) contactingbrain cells with a glutamate receptor antagonist and (B) determiningwhether the intracellular accumulation of amyloid is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. Modest internalization of Aβ in hippocampal slices treatedwith Aβ1-42 only.

FIG. 1A. Low magnification photomicrograph of a hippocampal explantshowing Aβ1-42 immunostaining is restricted to a subpopulation of cellsin CA1 stratum pyramidale (bracketed by arrows; DG, dentate gyrus).

FIG. 1B. Higher magnification photomicrograph of the field ofimmunostaining shows that Aβ-ir is localized within small puncta instratum pyramidale (sp) and, very faintly, within dendritic processes instratum radiatum (sr) (so, stratum oriens). The scale bar used in FIG.1B=0.5 mm for FIGS. 1A and 50 μm for FIG. 1B.

FIG. 2A-2D. Exogenous Aβ incorporation is enhanced by the integrinantagonist GRGDSP (SEQ. ID. No.3). Photomicrographs of sections from anexplant treated with Aβ1-42 plus 2 mM GRGDSP (SEQ. ID. No.3) for 6 daysand processed for the localization of Aβ-ir.

FIG. 2A Low magnification photomicrograph of a cultured slice showing abroad, dense field of Aβ-ir neurons which is confined to stratumpyramidale of fields CA1 and CA2, and the relatively greaterincorporation in this case as compared to the representative slicetreated with Aβ1-42 alone, shown in FIG. 1A.

FIG. 2B. Higher magnification image of CA1 stratum pyramidale showingincorporated Aβ-ir (“ir”=immunoreactive) is limited to neuronal cellbodies and proximal dendrites. Dark perinuclear cytoplasmic staining isevident.

FIG. 2C. High magnification photomicrograph of neurons in field CA1cshowing Aβ-ir is localized to puncta tightly clustered around thenucleus but also scattered into proximal apical and basal dendrites.Diffuse staining of the dendritic processes is also evident.

FIG. 2D. Photomicrograph of Aβ immunostaining at the border between CA1(left side) and CA2 (right side) stratum pyramidale of a slice treatedwith Aβ1-42 and GRGDSP (SEQ. ID. No.3) showing subfield differences inthe compartmentalization of sequestered amyloid peptide: relativelyhomogenous cytoplasmic immunostaining predominates in field CA2 whileCA1 pyramidal cells exhibit both diffuse cytoplasmic and denseperinuclear aggregates of Aβ-ir. The scale bar used in FIG. 2A=0.5 mm;50 μm in FIG. 2B; 24 μm in FIG. 4C-4D.

FIG. 3A-3F. Facilitation of Aβ internalization is specific to integrinantagonists. Photomicrographs of Aβ-ir in cultured hippocampal slicesincubated with Aβ1-42 in the presence of the inactive control peptideGRADSP (SEQ. ID. No.5), (FIG. 3A), the disintegrin echistatin or (FIG.3B) or the peptide integrin antagonist GRGDSP (SEQ. ID. No.3) (H,dentate gyrus hilus). Note that both antagonists of the RGD-bindingintegrins, GRGDSP (SEQ. ID. No.3) (FIG. 3C) and echistatin (FIG. 3B),increased the uptake of Aβ1-42 peptide and led to incorporation withinboth fields CA3 and CA1, whereas incorporation in the presence of GRADSP(SEQ. ID. No.5) (FIG. 3A) was modest and limited to the CA2 andsubicular boundaries of CA1 stratum pyramidale (FIG. 3A, arrows). PanelD shows Aβ1-42-ir at the border between fields CA1 and CA2 from a slicetreated with echistatin illustrating the clustering of Aβ-ir aroundnuclei in field CA1 and the more diffuse appearance of cytoplasmicstaining in pyramidal cells of field CA2: Similar regional differencesare seen in slices treated with GRGDSP (SEQ. ID. No.3). (E) Subicularneuron from an explant incubated with GRGDSP (SEQ. ID. No.3) and Aβ1-42showing the dense accumulation of Aβ-ir in granules around the nucleusand lighter labeling in swellings along the apical dendrite (small thinarrows) and axon (short arrow). (F) Aβ immunostaining of neurons in thedentate gyrus from a slice treated with GRGDSP (SEQ. ID. No.3) andAβ1-42 showing scattered labeled cells in CA3C stratum pyramidale, thecentral hilus (H) and the surrounding stratum granulosum (SG). The scalebar used in FIG. 3F=0.5 mm in FIG. 3A-3C; 50 μm in FIG. 3D; 18 μm inFIG. 3E and 80 μm in FIG. 3F.

FIG. 4A-4G. A survey and quantification of Aβ-1-42 uptake in fourtreatment groups; Aβ1-42 alone (FIG. 4A) or in the presence of GRADSP(SEQ. ID. No.5) (FIG. 4B), echistatin (FIG. 4C), or GRGDSP (SEQ. ID.No.3) (FIG. 4D).

Digitized images of Aβ-ir (black) in field CA1b in sections throughcultured hippocampal slices incubated with Aβ1-42 alone (FIG. 4A) or inthe presence of GRADSP (SEQ. ID. No.5) (FIG. 4B), echistatin (FIG. 4C),or GRGDSP (SEQ. ID. No.3) (FIG. 4D). Stained elements at or above aspecified and consistent density threshold were selected as “positiveparticles” and highlighted in black using the “density slice” functionof NIH Image software as described in Methods. Graphs in FIGS. 4E-4Gshow the mean area of selected Aβ-ir particles (FIG. 4E), the total areaof Aβ-ir particles (FIG. 4F), and the number of Aβ-ir particles (FIG.4G) within the sample field for slices treated with Aβ1-42 alone (Aβ),Aβ1-42 plus GRADSP (SEQ. ID. No.5) (RAD/Aβ), Aβ1-42 plus echistatin(Echi/Aβ) and Aβ1-42 plus GRGDSP (SEQ. ID. No.3) (RGD/Aβ). Values shownin FIG. 4E-4G are group means ±sem, expressed as a percent of valuesobtained in slices treated with Aβ alone; the “n” for each group isindicated in white on each bar. *p<0.05; **p<0.01; 2-tail t-test forcomparison to Aβ-alone values. The scale bar used in FIG. 4A=50 μm forto FIG. 4A-4D.

FIG. 5A-5C. Integrin antagonism potentiates amyloid effects on cathepsinD content. Hippocampal slices were cultured for 12 days followed by 6days incubation with (FIG. 5A) Aβ1-42 alone or (FIG. 5B-5C) Aβ1-42 plusGRGDSP (SEQ. ID. No.3) and then processed for the immunocytochemicallocalization of cathepsin D. Panels show CA1 stratum pyramidale. Intissue with the combined treatment (FIG. 5B-5C) cathepsin B-ir iselevated and present in coarser puncta as compared to staining in tissueexposed to Aβ1-42 alone (FIG. 5A). Arrows in B indicate some neuronswith cytoplasmic immunostaining and coarse immunoreactive inclusions.Arrowheads in FIG. 5C indicate cathepsin D-ir deposits within probablemicroglial cells. Insert in FIG. 5C shows intraneuronal cathepsin D-irpuncta that exhibit lysosome-like distributions. The scale bar used inFIG. 5A=50 μm for FIG. 5A-5C.

FIG. 6A-6D. Co-distribution of neuronal Aβ incorporation and reactivemicroglia. Photomicrographs of tissue from a hippocampal explant treatedwith Aβ1-42 and 2 mM GRGDSP (SEQ. ID. No.3) for 6 days and processed forthe simultaneous immunocytochemical localization of sequestered Aβ (darkgray) and ED-1 reactive microglia (black). (FIG. 6A) Low magnificationphotomicrograph showing that both Aβ-ir and ED-1-ir are clustered instratum pyramidale of CA1 and CA3c extending into the hilus (H); bothmarkers are low in the extra-hilar position of CA3 and stratumgranulosum. (FIG. 6B-6C) Higher magnification photomicrographs showdetails of co-distributed immunoreactivities within CA1b (FIG. 6B) andCA3c (FIG. 6C), two regions where Aβ uptake is most frequentlyencountered. (FIG. 6D) Photomicrograph of field CA2 showing the presenceof ED-1 positive microglia within a field of Aβ-ir (right side of panel)and the lack of reactive microglia in an immediately adjacent fieldlacking Aβ-ir neurons (left side of panel). The scale bar used in FIG.6D=0.5 mm for FIG. 6A; 50 μm for FIG. 6B; 80 μm for FIG. 6C and FIG. 6D.re co-localized.

FIG. 7. GRGDSP-enhancement (SEQ. ID. No.3) of Aβ uptake, cathepsin D-irincreases and microglial activation are all blocked by NMDA receptorantagonists. GRGDSP-enhancement (SEQ. ID. No.3) of Aβ uptake, cathepsinD-ir and microglial activation is blocked by the NMDA receptorantagonist AP5. Cultured hippocampal slices were treated with Aβ1-42 and2 mM GRGDSP (SEQ. ID. No.3) only (A,B,C) or in the presence of 50 μM AP5(A′, B′, C′) for 6 days and processed for the localization of Aβ1-42(A,A′), cathepsin D (B,B′), or ED-1 (C,C′) immunoreactivities. As shown,AP5 completely eliminated Aβ uptake (A′) and suppressed cathepsin D-ir(B′) and ED-1-ir (C′) to vehicle control levels. The scale bar used inFIG. 7C′=50 μm for all panels.

DEFINITIONS

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “A-beta” and “Aβ” refer to a peptide, also referred to as“amyloid beta peptide”, or “amyloid”, that is typically about 39-42amino acids in length. Aβ is a normally secreted proteolytic product ofthe carboxyterminal domain of the amyloid protein precursor (APP)(Busciglio, J. et al., Proc. Natl. Acad. Sci. USA 90:2092-2096 (1993),Haass, C et al., Nature 357:500-503 (1992), Shoji et al, Science258:126-129 (1992)). The 42 residue form (Aβ1-42) is generally agreed tobe the principal species in the senile plaques which constitute adiagnostic feature of Alzheimer's disease (AD) (Kang, J. et al., Nature325:733-736 (1987), Miller, D. L. et al., Arch. Biochem. Biophys.301:41-52 (1993)) and is preferentially generated over shorter forms(Aβ1-40) in genetic mutations related to familial AD (Haas, C. et al.,J. Biol. Chem. 269:17741-17748 (1994), Vigo, P. et al., J Neurochem61:1965-1968 (1993), Vincent, I. et al., Neurobiol. Aging 19:287-296(1998)). Transgenic mice that overexpress mutant APP gradually developplaques accompanied by neuropathology (see Sturchler, P. et al., Rev.Neurosci. 10:15-24 (1999)) with both effects being blocked byimmunization against Aβ(Motter, R. et al., Nature 400:173-177 (1999).The terms “A-beta” and “Aβ” are generically used to refer to an aminoacid length from 1-42 amino acids derived from, or modeled after, theAPP protein, and also the terms can refer to any homologs from human,rat, mouse, rabbit, guinea pig, etc., and their variants.

The term “activation” when used to refer to microglia may refer to atransformation of the microglia, for example, from a silent/quiet (slimcell body with ramified thin process) state to an active/macrophage-like(rounded cell body without process) state. Additionally, the term mayrefer to an enhanced ability to express and secrete cytokines.

“Alzheimer's disease” refers to a condition associated with: 1) theformation of neuritic plaques comprising amyloid beta protein andneurofibrillary tangles comprising tau proteins (primarily located inthe hippocampus and cerebral cortex) and, 2) an impairment in bothlearning and memory. “Alzheimer's disease” as used herein includes allkinds of Alzheimer's disease, including, e.g., early onset family typeAlzheimer's disease and late onset sporadic Alzheimer's disease.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group. Examples ofamino acid analogs include homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Amino acidmimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, butfunctions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

The terms “Apolipoprotein E” and “apoE” refers to a protein that isabout 299 amino acids in length and has a molecular weight of about34,000 Daltons, and plays a major role in lipid transport andmetabolism. Specifically, apoE functions as a cholesterol transportprotein within the periphery. ApoE is produced in abundance in brain andapoE-containing lipoproteins are the principal lipoproteins in theCerebro-Spinal Fluid (CSF). In the periphery, apoE expression isdramatically up-regulated in response to peripheral nerve injury. Asimilar role for apoE in the central nervous system (CNS) has beendescribed whereby apoE distributes cholesterol and phospholipids toneurons after injury. In normal rodent brain apoE is primarily localizedto glial cells, whereas in normal human brain apoE has been demonstratedin glia and neurons. After brain injury, intraneuronal apoE is markedlyincreased in both rodent and human brain. ApoE acts as a ligand forreceptors on neurons. The terms “apolipoprotein E” and “apoE” aregenerically used to refer to either apolipoprotein E protein or gene,and also the terms can refer to any homologs from rat, mouse, rabbit,guinea pig, etc., and their variants.

In humans, three common isoforms of apoE (i.e., apoE2, apoE3, and apoE4)are encoded by the different alleles 2, 3, and 4. The three differentapoE isoforms differ only by a single amino acid: apoE2 (cys112,cys158), apoE3 (cys112, arg158) and apoE4 (arg112, arg158). In vitrostudies indicate that the three apoE isoforms have differences.Especially, there is a difference in the ability of apoE3 and apoE4 tostimulate neurite outgrowth, bind to amyloid protein, bind tocytoskeletal proteins such as tau and microtubule associated proteinsand protect against oxidative stress. In general the apoE4 isoform has adetrimental effect when compared to the apoE3 isoform. For example, invitro experiments showed that apoE and apoE3 were able to bind tomicrotubules and form stable complexes with the microtubule associatedproteins tau and MAP2c while apoE4 was lacking this ability (Strimmatteret al., Exp. Neurol. 125:163-171 (1994)). Current evidence has alsoidentified the apoE4 allele as a major risk factor for sporadic andfamilial late-onset Alzheimer's disease as well as poor clinical outcomeafter certain forms of brain injury including that due to head traumaand spontaneous intracerebral hemorrhage. By contrast, possession of anapoE2 has been shown to protect against, or delay the onset of,Alzheimer's disease.

The term “apolipoprotein E4” or “apoE4” refers to apolipoprotein E4 orpolymorphic variants, alleles, interspecies homologs, or conservativelymodified variants thereof. The terms “apolipoprotein E4” and “apoE4” aregenerically used to refer to either apolipoprotein E4 protein or gene,as appropriate to the context. Preferably, apoE4 is from a mammal, e.g.,rat, mouse, human, rabbit, guinea pig, etc., and their variants. Thenucleotide and amino acid sequences of apoE4 are well-known in the art.For example, the human apoE4 gene is known and has the Genbank accessionnumber of M10065.

“Apolipoprotein E4 containing brain cells, or brain tissue containingthe same,” or “apoE4-containing brain cells, or brain tissue containingthe same,” refer to brain cells, or brain tissue containing the same,that can express apolipoprotein E4 proteins and/or contain the apoE4gene, as will be determined from the context. Typically,apoE4-containing brain cells, or brain tissue containing the same, arederived from a transgenic animal that comprises an exogenous apoE gene,e.g., a human apoE4 gene, polymorphic variants, alleles, interspecieshomologs, or conservatively modified thereof, which encode an apoE4protein. The methods for producing these transgenic animals arewell-known in the art and described in, e.g., U.S. Pat. No. 6,046,381.

“Brain cells” refers to cells and/or tissue containing the same. Braincells can be derived from any brain. For example, for use in the methodsof the invention, brain cells, or brain tissue containing the same, canbe those in or from a normal animal, an apoE-deficient animal, or anapoE4-containing animal. Preferably, brain cells, or brain tissuecontaining the same, are derived from a mammal, such as a rat, mouse,guinea pig, rabbit, etc. or transgenic animals with modulated levels ofneurofibrillary tangles, and/or tau proteins, and/or amyloid, and/oramyloid precursor proteins, and/or cathepsin D levels, and/or cysteineprotease levels, and/or mitogen activated kinases, and/or lysosomalenzyme levels, and/or cholesterol levels and/or altered cholesterolmetabolism, synthesis, storage, etc. The pathology modeling and drugtesting brain cell embodiments of the invention can be carried out inanimal models in vivo or in vitro. When provided in an embodiment inwhich the cells are cultured in vitro, unless otherwise indicated, thebrain cells, or brain tissue containing the same, can be provided in anyin vitro form capable of culture, for example, brain tissue thatcontains cells, or brain sections such as slices that contain cells,dissociated cells, cells bound to a solid support or in suspension, etc.

The term “compound that modulates integrins and/or integrin receptors”or “an agent(s) that modulate integrins or integrin receptors” refers tointer alia, a modulator or antagonist that may be selected from thegroup consisting of neutralizing and/or function blocking agents, suchas antibodies to integrin subunits alpha1, alpha2, alpha3, alpha4,alpha5, alpha6, alpha7, alpha8, beta1, beta2, beta3, beta4, beta5,beta6, beta7 and beta8. The compounds of the invention may be furtherselected from the group of peptides RGD, RGDS (SEQ. ID NO.1), GRGDS(SEQ. ID NO.2), GRGDSP (SEQ. ID NO.3), GRGDTP (SEQ. ID NO.4), mimeticsthereof and disintegrins such as echistatin found in snake venom.Preferably the peptides are soluble peptides. Also included are otherpeptides containing the RGD amino acid motif, and/or other peptidescapable of modulating integrin-mediated adhesion, or, alternativelyother, non-peptide entities, that are capable of modulating integrinsand/or integrin receptors and conservatively modified variants of saidpeptides. These compounds can be used individually or in a cocktailcontaining a combination of more than one compound. Sources for thepeptides include Calbiochem and Gibco or Life Technologies.

The term a “condition that modulates integrins or integrin receptors”refers to any condition that might accomplish integrin or integrinreceptor modulation. In addition to the compounds referred to in theearlier paragraph, additional examples of modulatory compounds includeamyloid beta peptide, oxidative free radicals (OH., O₂., etc.),lysosomal enzyme inhibitors(chloroquine,N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone,N-CBZ-L-phenylalanyl-L-phenyl-alanine-diazomethylketone,β-amyloid, and mimetics thereof, etc.), or inflammatory factors (TGFβ,IL-1β, LPS, etc.). These compounds can be used individually or in acocktail containing a combination of more than one compound or incombination with the above compounds.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants and allelesof the invention.

The following groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Serine (S), Threonine (T);    -   3) Aspartic acid (D), Glutamic acid (E);    -   4) Asparagine (N), Glutamine (Q);    -   5) Cysteine (C), Methionine (M);    -   6) Arginine (R), Lysine (K), Histidine (H);    -   7) Isoleucine (I), Leucine (L), Valine (V); and    -   8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).    -   (see, e.g., Creighton, Proteins (1984) for a discussion of amino        acid properties).

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Therecognized immunoglobulin genes include the kappa and lambda light chainconstant region genes, the alpha, gamma, delta, epsilon and mu heavychain constant region genes, and the myriad immunoglobulin variableregion genes. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well characterized fragments produced by digestion withvarious peptidases. This includes, e.g., Fab′ and F(ab)′2 fragments. Theterm “antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, or single chain antibodies. “Fc” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains, CH₁, CH₂ and CH₃, butdoes not include the heavy chain variable region.

Antibodies that specifically bind to Aβ can be prepared using anysuitable methods known in the art. See, e.g., Coligan, Current Protocolsin Immunology (1991); Harlow & Lane, supra; Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986); and Kohler &Milstein, Nature 256:495-497 (1975). Such techniques include antibodypreparation by selection of antibodies from libraries of recombinantantibodies in phage or similar vectors, as well as preparation ofpolyclonal and monoclonal antibodies by immunizing rabbits or mice (see,e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature341:544-546 (1989)). Specific polyclonal antisera and monoclonalantibodies will usually bind with a K_(d) of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 mM or better,and most preferably, 0.01 μM or better.

An “Aβ antibody” is an antibody or antibody fragment that specificallybinds to one or more epitopes found on Aβ1-42. Preferably, antibody thatspecifically binds to Aβ1-42 is used in embodiments of the invention,because this antibody recognizes and specifically binds to Aβ.

Examples of antibodies that may be used in the methods of the inventioninclude but are not limited to anti-alpha5, anti-alpha3 and anti-beta1,all of which are available from Chemicon.

“Brain cells” refer to cells and tissues obtained from any brain. Forexample, brain cells can be obtained from a normal animal, and/or atransgenic animal that comprises an altered endogenous apolipoprotein Egene (one or both alleles) that results in undetectable or significantlyless amount of apolipoprotein E proteins. Additionally, brain cells canbe obtained from a transgenic animal that comprises, for example, anexogenous apoE gene, e.g., a human apoE4 gene, polymorphic variants,alleles, interspecies homologs, or conservatively modified thereof,which encode an apoE4 protein. Preferably, brain cells are derived froma mammal, such as a rat, mouse, guinea pig, rabbit, etc. Unlessotherwise indicated, brain cells can be in the form of brain sectionssuch as slices, dissociated cells, etc. Alternatively, however braincells can be obtained from primary cell culture prepared from brains ofany of the above or established neuronal cell lines.

“Cathepsin D” is a lysosomal protease which typically exists in threeforms: the inactive proenzyme having an apparent molecular weight ofabout 55 kDa; the active single chain having an apparent molecularweight of about 50 kDa; and the active heavy chain having an apparentmolecular weight of about 38 kDa. This protease was previously shown tocleave tau protein at neutral (cytoplasmic) pH resulting in taufragments of approximately 29 kDa. See, e.g., Bednarski & Lynch, J.Neurochem. 67:1846-1855 (1996); Bednarski & Lynch, NeuroReport9:2089-2094 (1998).

The term “control” refers to the non-treated condition or substance.Control brain cells can be those that are not treated with a compound oragent that can modulate integrins and/or integrin receptors in the braincells. The term “control” can also refer to brain cells that are nottreated with a compound or agent that can interact with glutamatereceptors, such as NMDA-type glutamate receptors, in the brain cells. Insome embodiments, the term “control” can also refer to brain cells whichhave been treated with a compound or agent that can modulate integrinsand/or integrin receptors in the brain cells, but which have not beentreated with a compound or agent that can interact with glutamatereceptors, such as NMDA-type glutamate receptors, in the brain cells.

For example, when examining the effect of a compound to determine itsability to have an effect on one or more characteristics ofneurodegenerative disease, “control” brain cells could be brain cellsthat have not been treated with that compound, or brain cells assayed atthe beginning of the experiment (time=zero) before any compound-inducedchanges thereto, as will be clear from the context. In another example,as will be clear from the context, in some embodiments directed toapoE-deficient brain cells or apoE4-containing brain cells, the term“control” brain cells can also refer to normal brain cells (comprising awild-type or endogenous apolipoprotein E gene) which have been treatedwith a compound that increases an effective concentration of cathepsin Din the brain cells.

The term “deficient” refers to a decreased or lower amount of theindicated substance. For example, apolipoprotein E “deficient” braincells, or apoE “deficient” brain cells refer to brain cells that containless endogenous apolipoprotein E as compared to brain cells havingwild-type apolipoprotein E genes (for example, normal brain cells)measured or cultured under similar conditions. The term deficient mayalso refer to a variant that has an altered function, for example, braincells that are “deficient” in apoE may contain a variant of apoE thathas an altered function, e.g., in lipid transport, as compared towild-type apoE—such altered function not being able to substitute forthe unaltered function.

The term “effective,” as in an effective concentration of an “NMDAantagonist” refers to either an amount or an activity of the indicatedsubstance or condition that is sufficient to achieve the indicatedpurpose. For a first example, an effective concentration a “NMDAantagonist” would be one that produces positive results in terms ofalleviating the symptoms of a disease state having at least one ofseveral characteristics as described in this application. Positiveresults could be determined based on either altering the diseasecharacteristics or could be based on the use of brain imagingtechniques, electroencephalographic analysis or cognitive tests.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity orhigher over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

“Integrins” are cell surface receptors that mediate the physical andfunctional interactions between a cell and its surrounding extracellularmatrix (ECM). Classically, the role ascribed to integrins has been thatof an adhesion molecule, anchoring cells to the ECM. However, the morecontemporary spectrum of integrin function greatly exceeds that of merecell adhesion. Recent reports have demonstrated that the interactionbetween the ECM and cell surface integrins leads to intracellularsignaling events that affect cell migration, proliferation, andsurvival, which in the context of neoplastic cells, can translatedirectly into the malignant phenotype.

By “lysosomal dysfunction” is meant any activity, enzymatic ornon-enzymatic, or any property of the lysosomes that is affected in anegative manner relative to a control. This includes vesicle traffickingto or from lysosomes, the endocytic pathway, heterophagy or autophagy,and including the expression and activity of enzymes that are localizedin the lysosomes. By “inhibiting or suppressing a lysosomal function” ismeant lowering or decreasing one or more such activities from the levelor amount of such activity found in the non-inhibited or non-suppressedstate, including inhibiting or suppressing vesicle trafficking to orfrom lysosomes, and including inhibiting or suppressing the expressionor activity of a lysosomal enzyme. Such inhibition or suppression can beacute or chronic. Examples of lysosomal enzymes that can be inhibited orsuppressed include a lysosomal acid hydrolase, lysosomal protease,lysosomal nuclease, lysosomal lipase, amylase and a cathepsin. cathepsinB, cathepsin H or cathepsin L can be assayed using methods known in theart, for example, as described by Barrett, A. J. et al., Meth. Enzymol.80:535 (1981), Academic Press, New York, incorporated herein byreference.

Lysosomal dysfunction is further described as an abnormal lysosomalmorphology, chemistry or activity, which is detrimental to lysosomes orcells. Examples of lysosomal dysfunctions include a detrimental change,either increased or decreased, in the normal activity of the endocyticpathway, a detrimental change in lysosomal morphology, a detrimentalchange in the intra-lysosomal pH, and/or the activity(ies) of lysosomalenzyme(s).

“Neurodegenerative diseases” include almost all diseases in the centralnervous system accompanied by neuronal degeneration and include, e.g.,Alzheimer's disease, senile dementia, Parkinson's Disease, Huntingon'sDisease, frontotemporal dementias, frontotemporal dementia andParkinsonism, Pick's disease, Progressive supranuclear palsy pathology,etc. Neurodegenerative diseases also refers to pathologies and/ordisorders which are, in part, characterized by features typicallyassociated with human brain aging and related neurodegenerativediseases, such as Alzheimer's disease. Such characteristics includedepletion of synaptic proteins, meganeurite formation, induction ofneurofibrillary tangles, changes in lysosomal functions and chemistry,e.g., the proliferation of secondary lysosomes filled with lipofuscin,decreases in cathepsin L activities and increases in the levels ofcathepsin D, up-regulation and leakage of cathepsin D followed byphosphorylation of variants of tau fragments and accompanying tauproteolysis, activation of microglia, increased levels of sequestrationof and/or accumulation of and/or uptake of Aβ, etc. Accordingly,“neurodegenerative diseases” can include, e.g., an increased amount ofneurofibrillary tangles and/or lysosomes, the appearance of basophilicgranules in the mossy fiber terminal zone, the presence of secondarylysosomes with lipofuscin, amyloid deposition, amyloid plaques, neuriticplaques, synaptic loss, neuritic degeneration, neuronal death, increasedglial elements and/or increased glial activation (astrocytes,microglia), etc. Brain cells in accordance with embodiments of theinvention comprise increased levels of sequestration of and/oraccumulation of and/or uptake of Aβ, and/or evidence of lysosomaldysfunction, and/or microglia activation, but need not comprise all ofthese properties to be useful as a model of neurodegenerative diseases.Embodiments of the present invention are particularly useful as a modelof neurodegenerative diseases involving Aβ.

“Neurofibrillary tangles” refer to intraneuronal accumulations offilamentous material in the form of loops, coils or tangled masses.Neurofibrillary tangles seen in brain cells in vitro are sometimesreferred to herein as “tangle-like structures.” While neurofibrillarytangles can also be found during normal aging of the brain, they arefound in a significantly higher density in the brain of Alzheimer'sdisease patients, and other neurodegenerative diseases, such asprogressive supranuclear palsy, postencephalitic Parkinson disease,amylotrophic lateral sclerosis, etc. Robbins Pathologic Basis ofDisease, Cotran et al., 6th ed. (1999), p. 1330. They are commonly foundin cortical neurons, especially in the entorhinal cortex, as well as inother locations such as pyramidal cells of the hippocampus, theamygdala, the basal forebrain, and the raphe nuclei. Ultrastructurally,neurofibrillary tangles are composed predominantly of paired helicalfilaments (“PTHF”). A major component of PHF is an abnormallyhyperphosphorylated form of the protein tau and tau fragments.

The term “pharmaceutically effective amount” refers to an amountsufficient to alleviate, in any degree or manner, one or more of themanifestations or symptoms recognized or diagnosed as associated with amodifying disorder, modifying manifestations, or a modifying symptom.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

The term “reaction” when used to refer to microglial may refer to atransformation of the microglia, for example, from a silent/quiet (slimcell body with ramified thin process) state to an active/macrophage-like(rounded cell body without process) state. Additionally, the term mayrefer to an enhanced ability to express and secrete cytokines.

The term “sequestration of and/or accumulation of and/or uptake of Aβ”refers to a process, in part, whereby Aβ is physically accreted insideand/or outside of a brain cell. For example, Aβ is added to a mediumwhich comes in contact with brain cells, those contacted brain cells,through biological means, accrete the Aβ within their cell membranes,and/or within their lysosomal bodies, etc. Alternatively, the Aβcontaining medium can be contacted with brain tissue and the Aβ in thesolution can accrete outside the brain cells, and/or accrete to otherelements found outside the brain cells, such as elements of theextracellular matrix, etc.

The phrases “specifically binds to” or “specifically immunoreactivewith”, when referring to a binding moiety refers to a binding reactionwhich is determinative of the presence of a target antigen in thepresence of a heterogeneous population of proteins and other biologics.Binding moeities include any material capable of resolving the presenceof Aβ, such as antibody, dyes, silver, other contrast agents etc. Thus,under designated assay conditions, the specified binding moieties bindpreferentially to a particular target antigen and do not bind in asignificant amount to other components present in a test sample.Specific binding to a target antigen under such conditions may require abinding moiety that is selected for its specificity for a particulartarget antigen. A variety of immunoassay formats may be used to selectantibodies that are specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with anantigen. See Harlow and Lane (1988) Antibodies, A Laboratory Manual,Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically, a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background. Specific binding between anantibody or other binding agent and an antigen preferably has a bindingaffinity of at least 10⁶ M⁻¹. Preferred binding agents bind withaffinities of at least about 10⁷ M⁻¹, and preferably 10⁸ M⁻¹ to 10⁹ M⁻¹or 10¹⁰ M⁻¹.

“Transgenic animal” refers to a non-human animal that comprises anexogenous nucleic acid sequence present as an extrachromosomal elementor stably integrated in all or a portion of its cells, especially ingerm cells.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides novel methods for triggering brain cellsto induce the conditions of a brain afflicted with a neurodegenerativedisease and the brain cells produced by the method. In accordance withembodiments of the invention, brain cells are cultured in a medium whichmodulates integrins and/or integrin receptors in the brain cells, e.g.,by contacting the brain cells with a soluble peptide comprising theamino acid sequence RGD. The modulated integrins and/or integrinreceptors in the brain cells then trigger an increased amount ofsequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation. While some featuresof neurodegenerative diseases have been reproduced in other in vivo andin vitro models, some key features such as increased sequestration ofand/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation in the brain cells was oftenmissing in these models. The present invention advantageously providesbrain cells wherein the brain cells comprise, among other things,increased amounts of sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation.

In the present invention, any suitable brain cells are used. Preferably,brain cells are derived from a mammal, such as rat, mouse, guinea pig,rabbit, etc. Typically, brain cells are derived from normal animals. Incertain embodiments transgenic animals can be used, for example, atransgenic animal can be an apoE “knockout” animal, wherein one or bothalleles of the endogenous apoE gene is altered or ablated so that thebrain cells comprise undetectable or significantly less amount of apoEproteins. Alternatively, brain cells may be derived from transgenicanimals that comprise an apoE4 gene (e.g., a human apolipoprotein E4isoform and its homologs or conservatively modified variants thereof).Preferably, the endogenous apoE genes are completely or partly knockedout in these transgenic animals.

Brain cells, even without the treatment with a compound that modulatesintegrins and/or integrin receptors, have some residual amount ofsequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation. However, the levelsof sequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation in these untreatedbrain cells is too low to be regarded as an adequate model forneurodegenerative diseases, such as Alzheimer's disease.

Cathepsin D is a lysosomal protease which is found in the brain, alongwith other lysosomal proteases, such as cathepsin B and cathepsin L. Theactivities of these proteases change in the brain with aging. Forexample, the activity of cathepsin L decreases by up to 90% during brainaging, while the levels and activity of cathepsin D increase. SeeNakanishi et al., Exp. Neurol. 126:119-128 (1994). Moreover, theactivities of these cathepsin proteases are inter-related. For example,it was previously shown that inhibition of cathepsin B and L increasesprocathepsin D and its maturation into the active two-chain form(composed of heavy and light chain) within lysosomes. See Bednarski &Lynch, Neuroreport 9:2089-2094 (1998); Hoffman et al., Neurosci. Lett.250:75-78 (1998).

Surprisingly, when brain cells are contacted with a compound thatmodulates integrins and/or integrin receptors, increased sequestrationof and/or accumulation of and/or uptake of Aβ were induced inembodiments of the invention. Further, such treatment resulted inevident lysosomal dysfunction including increases in cathepsin D, andsuch treatments also resulted in the activation of microglia. Typically,the amount of increased sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activationseen in these treated brain cells is at least twice, sometimes at leastten times greater than the amount of sequestration of and/oraccumulation of and/or uptake of Aβ, and/or lysosomal dysfunction,and/or microglia activation seen in normal brain cells not treated withthe same compound. The density of sequestration of and/or accumulationof and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microgliaactivation in these brain cells treated with a compound that modulatesintegrins and/or integrin receptors is sufficiently high, mimicking thedensity of sequestration of and/or accumulation of and/or uptake of Aβ,and/or lysosomal dysfunction, and/or microglia activation typicallyfound in the brain of, for example, Alzheimer's disease patients. Sincebrain cells from transgenic animals contain many aspects and functionswhen compared to normal brain cells, transgenic brain cells, and braincells in vivo can also be used in embodiments of the invention.

Brain cells produced in accordance with the present invention have avariety of applications. For example, the brain cells can be used as anassay system to screen agents believed to modulate the amount ofsequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation. These agents can befurther tested in other systems and/or in vivo to confirm their efficacyin modulating the sequestration of and/or accumulation of and/or uptakeof Aβ, and/or lysosomal dysfunction, and/or microglia activation andpossibly other conditions and/or pathologies associated withneurodegenerative diseases, such as the cognitive decline seen inpersons afflicted with such disorders. In another example, the braincells can be used to study the morphological patterns of sequestrationof and/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation in the brain. In anotherexample, the brain cells can be used to study the effect ofsequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation in normal aging. Suchmorphological studies would provide additional information regarding thepathological process of aging and neurodegenerative diseases.

II. Production of Characteristics of Neurodegeneration

In one aspect, the invention provides brain cells, or brain tissuecontaining the same, and methods for producing brain cells, or braintissue containing the same, to a condition, or contacting brain cells,or brain tissue containing the same, with a compound that modulatesintegrins and/or integrin receptors to produce and/or increase thesequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation compared to a control(e.g., brain cells that are untreated with said compound(s)).

Embodiments of the invention include methods comprising:

(a) culturing brain cells; and

(b) contacting the brain cells with a compound that modulates integrinsand/or integrin receptors, thereby producing properties of an brainafflicted with a neurodegenerative disease, wherein the propertiesinclude increased sequestration of and/or accumulation of and/or uptakeof Aβ, and/or lysosomal dysfunction, and/or microglia activation, and/orrelated biochemical changes.

In some embodiments, a method for increasing sequestration of and/oraccumulation of and/or uptake of Aβ and/or lysosomal dysfunction, and/ormicroglia activation in brain cells comprises:

(a) culturing the brain cells in a medium which modulates integrinsand/or integrin receptors; and

(b) optionally, determining the production of and/or levels ofsequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation.

The brain cells produced in accordance with present methods mimic one ormore aspects of brain aging or the brain of patients with Alzheimer'sdisease or other neurodegenerative diseases, such as the presence ofincreased levels of sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation.However, brain cells produced by the present methods need not mimic allaspects of aged brain or neurodegenerative diseases to be useful as amodel of these conditions.

A. Sources of Brain Cells

Any suitable source of brain cells, or brain tissue containing the samecan be used in embodiments of the invention. Typically, brain cells, orbrain tissue containing the same are derived from a mammal, such as amouse, rat, guinea pig, rabbit, etc. Brain cells can be derived from anormal animal or other suitable transgenic animals. For example, apoEdeficient brain cells or apoE4 containing brain cells can be used inembodiments of the invention. A preferred embodiment includes in vivobrain cells.

Preferably, apoE deficient brain cells or apoE4 containing brain cellsare derived from transgenic animals that are genetically modified. Forexample, the brain cells can be derived from an apoE “knockout” animal,wherein the endogenous apoE gene in the genome has been altered orablated so that insubstantial or insignificant amount of apoE protein isproduced in the brain cells. For example, the function and/or expressionof the apoE protein in the apoE “knockout” animal is less than about30%, preferably less than about 10%, more preferably less than about 5%,still more preferably less than about 1%, compared to a normal animalwith the wildtype apoE genes. Most preferably, apoE deficient braincells are derived from apoE-knockout animals that have no apoE (i.e.,null) gene expression.

Typically, apoE4 containing brain cells can be derived from a transgenicanimal that comprises an exogenous apoE gene, e.g., a human apoE4 gene,polymorphic variants, interspecies homologs, or other conservativelymodified variants thereof. Preferably, in these transgenic animals thatcomprise an exogenous apoE4 gene, their endogenous apoE gene iscompletely or partly knocked out.

Transgenic animals comprising apoE deficient brain cells can be producedby recombinant methods known in the art. For example, the endogenousapoE gene function can be altered or ablated by, e.g., the deletion ofall or part of the coding sequence, or insertion of a sequence, orsubstitution of a stop codon. In another example, the non-codingsequence of the apoE gene in the chromosome can be modified by, e.g.,deleting the promoter region, the 3′ regulatory sequences, enhancersand/or other regulatory sequences of the apoE gene in the chromosome. Inyet another example, apoE deficient transgenic animals can be producedby introducing an anti-sense construct that blocks the expression of theendogenous apoE gene products. In some cases, it may be desirable toproduce conditional “knock-out” transgenic animals, wherein thealteration in the apoE gene can be induced by, e.g., exposure of theanimal to a substance that promotes the apoE gene alterationpostnatally. Preferably, both alleles of the apoE gene in the chromosomeare altered in these transgenic animals.

The methods for producing transgenic animals are well known anddescribed in, e.g., U.S. Pat. Nos. 5,464,764, and 5,627,059, thedisclosures of which are incorporated herein by reference. Inparticular, the following references describe methods for producingapoE-deficient homozygous rodents: Plump et al., Cell 71:343-353 (1992);and Gordon et al., Neuroscience Letters 199:1-4 (1995), the disclosuresof which are incorporated herein by reference. Moreover, some apoEdeficient transgenic animals are commercially available. For example,apoE-deficient homozygous nice, such as C57B1/6J-Apoetm1Unc strain, areavailable from the Jackson laboratory, Bar Harbor, Me.

Moreover, apoE4 containing brain cells can be derived from a transgenicanimal that comprises an exogenous apoE gene. For example, an exogenousapoE gene can be a human apoE4 gene, its interspecies homologs,polymorphic variants, or conservatively modified variants thereof. Inhuman, three isoforms (apoE2, apoE3 and apoE4) express variants of apoE.Among these isoforms, apoE4 is known in the art to encode an apoEprotein that is deficient in various functions. For example, compared toapoE3 that stimulates neurite extension, apoE4 was shown to inhibitneurite extension. Nathan et al., Soc. Neurosci. 20(Part 2):1033 (1994).It has also been suggested that in vitro tau interacts with apoE3, butnot with apoE4. Stritmatter et al., Exp. Neurol. 125:163-171 (1994).Moreover, the human apoE4 isoform has been described as a risk factor ofAlzheimer's disease (see, e.g., Peterson et al., JAMA 273:1274-1278(1995)). Since brain cells comprising an apoE4 gene appear to lack manynormal functions that other apoE isoforms possess, like the apoEdeficient brain cells, transgenic animals that comprise an apoE gene orits variants may also be used as a source of brain cells in embodimentsof the invention.

Such transgenic animals can be produced using various apoE nucleotidesequences known in the art or conservatively modified variants thereof.For example, the human apoE4 gene has the Genbank accession numberM10065. The mouse apoE gene has the Genbank accession number D00466.Other homologs or polymorphic variants of apoE genes can also be readilyidentified. For example, homologs or polymorphic variants of a knownapoE gene can be isolated using nucleic acid probes by screeninglibraries under stringent hybridization conditions. Exemplary stringenthybridization conditions are as follows: a hybridization in a buffercontaining 50% formamide, 5×SSC, and 1% SDS, at 42° C., or 5×SSC, 1%SDS, at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. In somecases, moderately stringent conditions may be used to clone homologs orpolymorphic variants of a known apoE gene. An example of a moderatelystringent condition includes a hybridization in a buffer of 40%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. Thesource of homologs can be any species, e.g., rodents, primates, bovines,canines, human, etc.

In some embodiments, it may be desirable to use modified or mutatedversions of apoE genes. For example, a modified version of a human apoE4gene, when introduced into a transgenic animal, may be capable ofproducing a higher density of neurofibrillary tangles compared to theunmodified human apoE4 gene. Techniques for in vitro mutagenesis ofcloned genes are well-known in the art and can be readily applied formaking a modified or mutated apoE gene. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, CSH Press (1989). The functionaleffect of a modified or mutated apoE gene can be further tested in vivoor in vitro. For example, a transgenic animal comprising a modified ormutated apoE gene can be produced using the methods known in the art.The change in the properties in apoE brain cells (e.g., theneurofibrillary tangle or phosphorylated tau fragment production) can bedetermined using the methods described below.

Methods for producing a transgenic animal comprising an exogenous apoEgene are known. Generally, an exogenous apoE gene, such as the humanapoE4 gene or its variant, is operatively linked to any suitableregulatory element for expressing the apoE4 gene. Preferably, theexogenous apoE gene is operably linked with a mammalian apoE promoter,such as human apoE4 regulatory sequences. This construct can beintroduced into an animal using methods known in the art. In thesetransgenic animals comprising an exogenous apoE gene (e.g., human apoE4gene), preferably the endogenous apoE gene is partially or completelyknocked out so that the endogenous apoE expression or function isinsubstantial. Moreover, methods for producing transgenic animalscomprising various human apoE isoforms are described in, e.g., U.S. Pat.No. 6,046,381 and U.S. Pat. No. 5,767,337, the disclosure of which areherein incorporated by reference.

B. Culturing Brain Cells

Brain cells derived from animals described herein can be processed inany suitable manner. For example, the brain can be processed in the formof tissue sections, such as brain slices. Alternatively, the braintissues can be processed in the form of dissociated cells. Whether inthe form of brain slices, dissociated cells, or other forms, they willbe generically referred to as “brain cells” herein, unless otherwiseindicated.

Additionally, various cell lines may be used in the methods of theinvention including integrin knockout cell lines (Matter et al., J.Cell. Biol. 141:1019-1030, 1998), human neuroblastoma cell line (IMR-32)(ATCC, Manassas, Va.), CHO and NMDA receptor knockout cells such asNR1−/− and NR2−/−.

In one embodiment, an in vivo model is used. Such in vivo models have anadvantage in that they retain the native brain architecture andenvironment. The effects that are brought about by the methods of theinvention are presented against the background of a physiologicalenvironment that is more likely to mimic such conditions in humans. Invivo models are also more amenable to long term analysis than areprimary cultures, or brain slice cultures. Another advantage is thatmultiple samples can be taken at the same time from the same animal andfrom different parts of the brain.

Preferably, the brain is processed in the form of brain slices so thatneuronal circuitry or other biological functions are maintained. Asuitable thickness of the brain slice is readily determinable by thoseof skill in the art, and may be varied depending on the culturecondition or subsequent analysis methods. For example, the brain can besliced in the thickness of about 200 μm to about 800 μm, preferablyabout 350 μm to about 400 μm. The entire brain or portions of the braincan be processed into slices. For example, suitable brain slices mayinclude a hippocampal slice, an entorhinal cortex slice, anentorhinohippocampal slice, a neocortex slice, a hypothalamic slice, ora cortex slice. Since Aβ accumulations tend to develop more prominentlyin the hippocampal region, a hippocampal slice is preferably used.

Alternatively, the brain can be processed into dissociated brain cells.The entire brain or selected regions of the brain (e.g., the hippocampalregion) can be dissociated and maintained in a culture. Generally, thebrain tissue is dissected, minced and digested in an enzyme (e.g.,trypsin) for a suitable period of time. Then cells are centrifuged andplated at a low density in culture plates. The methods for dissociatingcells are well-known in the art. See, e.g., Freshney, Culture of AnimalCells a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994),incorporated herein by reference.

Brain cells in the form of slices or dissociated cells can be maintainedin culture. Suitable culture conditions for brain cells are well-knownin the art. For example, brain cells can be placed onto culture plates,preferably on a support, such as a matrix or membrane, which allowscells to attach. Any suitable medium can be used in maintaining theculture of brain cells. Typically, the culture of brain cells ismaintained in a medium that has all the essential nutrients. The culturemedium generally has a neutral pH, e.g., between about pH 7.2 to about7.8, and is maintained at a temperature between about 4° C. to about 40°C., typically at about 37° C. The culture of brain cells is typicallymaintained in an atmosphere that contains CO₂, preferably at 5% CO₂. Ingeneral, the culture can be maintained for at least about 60 days withaperiodic replacement of culture medium.

C. Treatment of Brain Cells with an Agent Capable of ModulatingIntegrins and/or Integrin Receptors to Trigger the Sequestration ofand/or Accumulation of and/or Uptake of Aβ.

To increase the sequestration of and/or accumulation of and/or uptake ofAβ, and/or lysosomal dysfunction, and/or microglia activation, braincells are contacted with a compound that modulates integrins and/orintegrin receptors (“integrin antagonist”). Preferably, a integrinantagonist increases the sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activationin brain cells by at least about 30%, preferably at least about 50%,more preferably at least about 80%, most preferably at least about 100%,compared to a control (e.g., brain cells untreated with the compound).

Any suitable integrin antagonist compound can be used in embodiments ofthe invention. The modulator or antagonist may be selected from thegroup consisting of neutralizing and/or function blocking antibodies forintegrin subunits alpha1, alpha2, alpha3, alpha4, alpha5, alpha6,alpha7, alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 andbeta8. The methods of the invention are further drawn to a peptideselected from the group of peptides consisting of RGD, RGDS (SEQ. IDNO.1), GRGDS (SEQ. ID NO.2), GRGDSP (SEQ. ID NO.3), GRGDTP (SEQ. IDNO.4), mimetics thereof and disintegrins such as echistatin found insnake venom. Disintegrins block integrin-mediated events in a widevariety of circumstances (Huang, T. F., Cell Mol Life Sci 54:527-540(1998)). Examples of disintegrins include echistatin and triflavin thatcan be obtained from Sigma.

Other suitable integrin antagonist compounds, and/or agents whichmodulate integrins and/or integrin receptors are readily determinable bythose skilled in the art. For example, a test compound can be contactedwith brain cells and/or brain membranes, integrins, integrin receptors,etc. Then the activity or the amount of integrin antagonism can bemeasured.

The activity or the amount of integrin antagonism is then compared witha control amount (e.g., the amount of integrin-mediated adhesionobserved when the assay system is not treated with the test compound). Atest compound is referred to as a “integrin antagonist” if it modulatesthe activity of any one or more of integrins, integrin receptors, etc.)by, e.g., at least about 30%, preferably at least about 50%, morepreferably at least about 80%, most preferably at least about 100%,compared to a control.

Brain cells can be contacted with an integrin antagonist compound at anysuitable time. For example, brain cells can be contacted with anintegrin antagonist compound when the culture is first established, orat a later time after maintaining the culture for a few days.Preferably, brain cells are contacted with an integrin antagonistcompound for a period of 1-18 days, preferably for a period of 2-4 days.To induce the sequestration of and/or accumulation of and/or uptake ofAβ and/or lysosomal dysfunction, and/or microglia activation, anintegrin antagonist compound is typically added at a concentration of 50μM to about 5000 μM, more typically at a concentration of about 1 mM toabout 3 mM.

Other modulatory compounds, in addition to an integrin antagonistcompound(s), can be added in the culture medium to further facilitatethe sequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation or otherneurodegenerative features in brain cells. Examples of modulatorycompounds include oxidative free radicals (Fe³⁺, H₂O₂, etc.), lysosomalenzyme inhibitors (chloroquine,N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone,N-CBZ-L-phenylalanyl-L-phenylalanine-diazomethylketone,and mimetics thereof, etc.), or inflammatory factors (TGFb, IL-1b, LPS,etc.).

Typically, brain cells in a culture are treated with an integrinantagonist under a condition such that the amount of sequestration ofand/or accumulation of and/or uptake of Aβ and/or lysosomal dysfunction,and/or microglia activation is increased by at least about 10%, or atleast about 20%, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 80%, or at least about 100%, or atleast about 150%, or at least about 200%, compared to a control (e.g.,brain cells that are cultured in substantially the same condition butwithout an integrin antagonist compound(s)). Also, brain cells that aretreated with an integrin antagonist compound generally produce thesequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation at a significantlyhigher level, typically at least two times, sometimes ten times, morethan normal brain cells treated with the same compound. Preferably, thetreatment conditions (e.g., concentration of integrin antagonistcompound(s), the period of incubation, etc.) are selected so that thesequestration of and/or accumulation of and/or uptake of Aβ and/orlysosomal dysfunction, and/or microglia activation produced in treatedbrain cells is similar to the density of these materials in the brainsof patients with Alzheimer's disease, the aging brain, and/or otherneurodegenerative diseases.

D. Determining the Sequestration of and/or Accumulation of and/or Uptakeof Aβ.

After treating brain cells with an integrin antagonist compound, thelevels of sequestration of and/or accumulation of and/or uptake of Aβ,and/or lysosomal dysfunction, and/or microglia activation can bedetermined if desired. Determination of the sequestration of and/oraccumulation of and/or uptake of Aβ, and/or lysosomal dysfunction,and/or microglia activation can be qualitative or quantitative. In someapplications, it may be sufficient to visually inspect the sequestrationof and/or accumulation of and/or uptake of A-β, and/or lysosomaldysfunction, and/or microglia activation. For example, it may be usefulto visually observe the timing and the pattern of sequestration ofand/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation at different regions of thebrain. In other applications, it may be desirable to quantitate thesequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation. Quantitation wouldbe particularly useful in a screening assay for agents that modulate thesequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation.

Any suitable methods known in the art can be used to determine thelevels of sequestration of and/or accumulation of and/or uptake of Aβ,and/or lysosomal dysfunction, and/or microglia activation. For example,brain cells, in the form of brain slices, dissociated cells, or othersuitable forms, can be stained using conventional staining methods. Forexample, the brain cells can be fixed and stained with a silver stain(Bielschowsky) (Bancroft et al., Theory and Practice of HistologicalTechniques. New York, Churchill Livingstone, Edinburgh, 1996) stain ortoluidine blue. Then the stained Aβ, lysosomes, cathepsin D, and/ormicroglia elements can be visualized by microscopy.

In another example, the brain cells can be stained by immunostaining,and the sequestration of and/or accumulation of and/or uptake of Aβ,and/or lysosomal dysfunction, and/or microglia activation can bevisualized. In immunostaining, suitable capture reagents, such asantibodies that specifically bind to Aβ, lysosomes, cathepsin D, and/ormicroglia elements, can be used. Preferably, antibodies preferentiallybind to Aβ, lysosomes, cathepsin D, and/or microglia elements and do notsignificantly cross-react with other proteins in the brain cells. Forexample, the antibodies that specifically bind to Aβ, lysosomes,cathepsin D, and/or microglia elements have less than 50%, preferablyless than 30%, more preferably less than 10% crossreactivity with otherantigens in the brain tissue.

Examples of antibodies that preferentially bind Aβ, lysosomes, cathepsinD, and/or microglia elements include antibodies anti-Aβ1-42, Ab-2 fromCalbiochem, ED-1, and other known to those skilled in the art.Preferably, anti-Aβ1-42 is used in embodiments of the invention to bindAβ, and Ab-2 is used in embodiments of the invention to bind cathepsinD, while ED-1 is used in embodiments of the invention to identifyactivated microglia.

In immunostaining, an antibody against Aβ, and/or cathepsin D, and/orlysosomes and/or microglia elements is added to brain cells, and thebrain cells are incubated for a sufficient time to allow binding betweenthe antibody and Aβ, and/or cathepsin D, and/or microglia elements. Theantibody may be labeled with a variety of labels that are detectable.Useful labels include magnetic beads (e.g., DYNABEADS™), fluorescentdyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase), and calorimetric labelssuch as colloidal gold or colored glass or plastic beads (e.g.,polystyrene, polypropylene, latex, etc.). Alternatively, the antibodymay be unlabeled, and a label may be coupled indirectly. For example, anunlabeled primary antibody can be added to the culture to bind Aβ,and/or cathepsin D, and/or lysosomes and/or microglia elements, and thena labeled secondary antibody can be used to amplify the signal fordetection.

Means of detecting labels are well known to those of skill in the art.For example, where the label is a radioactive label, means for detectioninclude a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Simple calorimetric labels maybe detected simply by observing the color associated with the label.

Alternatively, the levels of Aβ, and/or cathepsin D, and/or lysosomesand/or microglia elements can be determined using cell lysate in animmunoassay. An immunoassay can be performed in any of several formats.These formats include, for example, an enzyme immune assay (EIA) such asenzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), aWestern blot assay, or a slot blot assay. For a review of the generalimmunoassays, see, e.g., Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). A general overview of applicabletechnology can also be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988). See, also, U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168.

In one embodiment, an immunoblotting can be used to quantify the amountof Aβ, and/or cathepsin D, and/or lysosomes and/or microglia elementsproduced in brain cells treated with an integrin antagonist compound.Generally, brain cells are disrupted in an electrophoresis sample bufferand are treated to obtain a fraction that contains proteins. Theproteins are separated by gel electrophoresis and transferred to amembrane that binds the proteins nonspecifically. The location of Aβ,and/or cathepsin D, and/or lysosomes and/or microglia elements on themembrane is determined using, e.g., a labeled primary antibody or anunlabeled primary antibody, followed by a labeled secondary antibody. Adetectable label may be, e.g., a radio-label or a fluorescent label or,an enzyme label. Then the membrane comprising a detectable label can bescanned, and digitized images can be quantitatively analyzed bydensitometry.

In another embodiment, a sandwich assay can be performed by preparing abrain cell lysate sample, and placing the sample in contact with a solidsupport on which is immobilized a plurality of antibodies that bind Aβ,and/or cathepsin D, and/or lysosomes, and/or microglia elements. Thesolid support is then contacted with detection reagents for Aβ, and/orcathepsin D, and/or lysososmes, and/or microglia elements. Afterincubation of the detection reagents for a sufficient time to bind asubstantial portion of the immobilized Aβ, and/or lysososmes, cathepsinD, and/or lysosomes, and/or microglia elements, any unbound labeledreagents are removed. The detectable label associated with the detectionreagents is then detected. For example, in the case of an enzyme used asa detectable label, a substrate for the enzyme that turns a visiblecolor upon action of the enzyme is placed in contact with the bounddetection reagent. A visible color will then be observed in proportionto the amount of Aβ, and/or cathepsin D, and/or lysosomes and/ormicroglia elements in the sample.

The above described detection methods are merely exemplary, and othersuitable detection methods will be apparent and can be readilysubstituted by one of skill in the art.

III. Screening Assays

In another aspect, the invention provides methods for screening agentsthat modulate the sequestration of and/or accumulation of and/or uptakeof Aβ, and/or lysosomal dysfunction, and/or microglia activation thatare induced by an integrin antagonist compound in brain cells.Particularly useful agents include those that are capable of inhibitingthe sequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglial activation in the brain cells.

Generally, screening methods comprise:

(a) contacting brain cells with an integrin antagonist compound thatmodulates integrins and/or integrin receptors in the brain cells,wherein the modulated integrins and/or integrin receptors are capable ofincreasing the amount of sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activationin the brain cells;

(b) contacting the brain cells with an agent; and

(c) determining whether the agent modulates the amount of sequestrationof and/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation in the brain cells treated withthe agent compared to the brain cells that are not treated with theagent.

To produce brain cells comprising increased levels of sequestration ofand/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation, the methods described insection II above can be used, and these methods will not be repeated inthis section. Typically, brain cells in the form of slices arepreferably used in the screening assays, since the neuronal circuitryand other biological functions are more intact in brain slices, comparedto dissociated brain cells, allowing the brain slices to better mimicthe physiological condition of the brain. Preferably, the amounts and/oractivities of integrin antagonist compound(s) and other cultureconditions are adjusted so that the levels of sequestration of and/oraccumulation of and/or uptake of Aβ, and/or lysosomal dysfunction,and/or microglia activation in the brain cells (prior to contacting withan agent) is similar to the density of these materials found in thebrains of persons suffering form neurodegenerative diseases, such asAlzheimer's disease.

To screen agents that modulate the sequestration of and/or accumulationof and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microgliaactivation, brain cells are contacted with a test agent. An “agent” or“substance” refers to any molecule, including, e.g., a chemical compound(organic or inorganic), or a biological entity, such as a protein,sugar, nucleic acid or lipid, that modulates the amount of sequestrationof and/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation in brain cells. Generally, atest agent or substance is added to the culture medium in the range from0.1 nM to 10 mM.

Agents can be obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, libraries ofnatural compounds in the form of bacterial, fungal, plant and animalextracts can be tested. Known pharmacological agents may be subjected todirected or random chemical modifications, e.g., alkylation,esterification, amidification, etc. to produce a library of structuralanalogs. Alternatively, a library of randomly or directed synthesizedorganic compounds or biomolecules (e.g., oligonucleotides andoligopeptides) can be used as a source of agents. Preparation andscreening of combinatorial libraries are well known to those of skill inthe art. See, e.g., U.S. Pat. No. 5,010,175, PCT Publication No. WO93/20242, PCT Publication No. WO 92/00091, Chen et al., J. Amer. Chem.Soc. 116:2661 (1994), U.S. Pat. No. 5,539,083.

Since the levels of sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activationis correlated with integrin and/or integrin receptor modulation in braincells, an integrin antagonist may be effective in increasing thesequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation or other relatedneuropathological lesions. Accordingly, a library of putative integrinand/or integrin receptor modulators can be used as a source of agents ina screening assay. Methods for producing a library of potential integrinand/or integrin receptor modulators are known. For example, acombinatorial library of agents against the active site of integrinsand/or integrin receptors can be synthesized by one skilled in the art.A library of such agents can be screened by methods in accordance withembodiments of the invention.

An agent can be contacted with brain cells at any suitable time. Forexample, an agent can be contacted with brain cells prior to contactingthe brain cells with an integrin antagonist compound. In anotherexample, the brain cells can be contacted with the agent after the braincells are contacted with an integrin antagonist compound. Preferably,the brain cells can be contacted simultaneously with the integrinantagonist compound and the agent. Generally, brain cells are contactedwith an agent for a period of time sufficient to allow the agent to takean effect. Typically, the brain cells and an agent are contacted for aperiod of between about 1 minute to about 30 days, preferably betweenabout 30 minutes to about 6 days. Typically, during this time, theculture of brain cells is maintained at a temperature between about 4°C. to about 40° C., preferably at 37° C., at atmosphere containing about0 to 10% CO₂. Other suitable experimental conditions are readilydeterminable by those skilled in the art.

A number of assays known in the art can be used to determine the effectof candidate agents on the sequestration of and/or accumulation ofand/or uptake of Aβ, and/or lysosomal dysfunction, and/or microgliaactivation in brain cells. For example, various staining or immunoassaysdescribed above can be used, and the details of these assay techniqueswill not be repeated in this section. Other suitable assays will bereadily determinable by those of skill in the art, and can be applied indetecting the sequestration of and/or accumulation of and/or uptake ofAβ, and/or lysosomal dysfunction, and/or microglia activation.

In determining whether an agent modulates the integrin antagonistinduced sequestration of and/or accumulation of and/or uptake of Aβ,and/or lysosomal dysfunction, and/or microglia activation in braincells, experiments are typically carried out with a control. A controlcan be, e.g., adding no agent or adding a different amount or type ofagent is added and extrapolating and determining the zero amount. Astatistically significant difference in a test amount (e.g., brain cellstreated with a test agent) and a control amount (e.g., brain cellsuntreated with a test agent) of sequestration of and/or accumulation ofand/or uptake of Aβ, and/or lysosomal dysfunction, and/or microgliaactivation indicates that the test agent modulates the sequestration ofand/or accumulation of and/or uptake of Aβ, and/or lysosomaldysfunction, and/or microglia activation. For example, inhibition ofsequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation is achieved when thetest amount of sequestration of and/or accumulation of and/or uptake ofAβ, and/or lysosomal dysfunction, and/or microglia activation relativeto the control amount is about 90% (e.g., 10% less than the control),optionally 80% or less, 70% or less, 60% or less, 50% or less, 40% orless, or 25-0%.

Brain cells in accordance with embodiments of the invention provide anin vitro model for neurodegenerative diseases, such as Alzheimer'sdisease, and brain aging. As such, brain cells contacted with integrinantagonists provide a cost and time efficient in vitro model to studysuch diseases. For example, brain cells produced in accordance withembodiments of the invention can be advantageously used to screen agentsthat may modulate the sequestration of and/or accumulation of and/oruptake of Aβ, and/or lysosomal dysfunction, and/or microglia activationin the brain cells. Efficacious agents that are identified by in vitroscreening methods described herein can be further tested to determinetheir efficacy in vivo. Some of these agents can potentially be usefulas therapeutic compounds for neurodegenerative diseases, includingAlzheimer's disease.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE 1 I. Materials and Methods

A. Preparation and Maintenance of Hippocampal Slice Cultures

Organotypic hippocampal cultures were prepared using the technique ofStoppini et al. (1991). Briefly, hippocampi were harvested from brainsof 9-12 days old Sprague-Dawley rat pups under sterile conditions.Sections were cut (400 μm thick) perpendicular to the long axis ofhippocampus using a McIllwain tissue chopper and were collected into acutting medium consisting of MEM with Earle's salts (Gibco, RockvilleMd.), 25 mM HEPES, 10 mM Tris base, 10 mM glucose, and 3 mM MgCl2 (pH7.2). Slices were positioned onto 30 mm cell culture inserts(Millicell-CM, Millipore, Bedford, Mass.) that were placed in 6 wellculture trays with 1 ml of growth medium per well [growth medium: MEMwith Hank's salts (Gibco), 20% horse serum, 3 mM glutamine, 25 mM HEPES,5 mM NaHCO3, 25 mM glucose, 0.5 mM ascorbate, 2 mM CaCl2, 2.5 mM MgCl2,0.5 mg/l insulin, and penicillin, pH 7.2]. The cultures were incubatedat 35° C. with a 5% CO2-enriched atmosphere and the media was changedevery other day until use 10-12 days later.

B. Treatment with Aβ and Integrin Antagonist

Cultured hippocampal slices were exposed to media containing the humanAβ1-42 sequence in the presence or absence of the integrin antagonistpeptide, Gly-Arg-Gly-Asp-Ser-Pro, or GRGDSP (SEQ. ID. No.3) (2 mMdissolved in media) (Ruoslahti, E., Ann Rev Cell Dev Biol 12:697-715(1996)). In some cases, slices were co-treated with the specific NMDAreceptor antagonist 2-amino-5-phosphonovalerate (AP5) (Sigma) at 50 μM.Control slices in neighboring wells received media-vehicle only. Aβ1-42peptide solution was freshly prepared before the start of the experimentusing 0.1N NaOH, pH adjusted with 0.1N HCl to 7.4, and then diluted withserum-free culture medium. Aβ was applied at 30 μM for 8-10 hr afterwhich it was diluted to 15 μM by the addition of fresh culture mediacontaining heated treated horse serum. Treatment was repeated everyother day for a total 6 days. Disintegrins are small (4-10 kDa)RGD-containing, cysteine-rich peptides from snake venom that bindintegrins with high affinity (Kd˜1 nM-0.1 μM) and are potent antagonistsof integrin functions (Huang, T. F., Cell Mol Life Sci 54:527-540 (1998)for review). In some experiments the disintegrin echistatin (2 μMdissolved in media) was applied instead of GRGDSP (SEQ. ID. No.3). Tofurther test the specificity of the integrin antagonists, the inactivecontrol peptide Gly-Arg-Ala-Asp-Ser-Pro (GRADSP) (Torimoto, Y., et al.,J Exp Med 172:1315-1323 (1990)) was used at 2 mM.

C. Immunocytochemistry

The following treatment, slices were thoroughly washed with 0.1M sodiumphosphate buffered saline (PBS), fixed for 12-16 hr in cold 0.1Mphosphate buffer (PB; pH 7.2) containing 4% paraformaldehyde,cryoprotected in 20% sucrose/PB for 1-2 hr and then carefully removedfrom the insert membranes. Serial sections were cut at 25 μm thick,parallel to the broad face of the explant, using a freezing microtome.Immunocytochemistry was performed using the standard avidin biotinhorseradish peroxidase complex (ABC) method using reagents andinstructions of the VECTASTAIN® Elite ABC kit from Vector Laboratories(Burlingame, Calif.) with diaminobenzidine tetrahydrochloride (0.05% in50 mM Tris-HCl buffer, pH 7.4) as chromagen and PBS for rinses and asdiluent for antibody incubations. Briefly, free-floating sections werepreincubated with 3% normal goat serum (for anti-Aβ1-42 andanti-cathepsin D antibodies) or 10% normal horse serum (for ED-1) in PBSfor 1 hr at room temperature. Sections were then incubated withanti-human Aβ1-42 (1:5000, gift from Dr. C. Glabe, UCI; specific for thehuman Aβ1-42 sequence; does not recognize rat amyloid peptides),anti-cathepsin D (1:100, Calbiochem, San Diego) antisera in 1.5% normalgoat serum or monoclonal antibody ED-1 (1:1000; Serotec, Oxford) in 5%normal horse serum overnight at 4° C. Sections were washed in PBS,incubated in biotinylated anti-rabbit or anti-mouse IgG (both at 1:200)for 2-3 hr, washed in PBS, incubated in the avidin-biotin complexsolution for 45 min and then processed through the diaminobenzidinereaction. After final rinses in PBS, sections were mounted on SuperFrostPlus slides (Fisher Scientific, Pittsburgh), air-dried, dehydrated in aseries of graded ethanols, and coverslipped from Clearing Solvent(Stephens Scientific, Kalamazoo, Mich.) with Permount (Fisher).

In some cases a dual-chromogen procedure was used for simultaneousimmunolabeling of Aβ1-42 and the microglial antigen ED-1. Briefly,sections were first immunostained with anti-A 1-42 usingdiaminobenzidine tetrahydrochloride as the chromagen (as above) to yielda dark grey reaction product within the labeled neurons. The sectionswere then washed with PBS, and immunostained for ED-1 following the samesteps up to the chromagen reaction. At this point, the sections wereprocessed through several changes of 0.02 M PB, pH 6.5, to lower the pHand ionic strength, and were then incubated with 0.005% benzidinedihydrochloride and 0.001% hydrogen peroxide to yield granularblue-black deposits.

To control for potential non-specific labeling in immunostainingprocedures, tissue was processed through all steps but with the primaryantisera replaced by PBS or normal sera in the first incubation. Nocellular or regional labeling was observed under these conditions.

Photomicrographs were obtained using an Olympus AX70 microscope andKodak PlusX film (Kodak, Rochester, N.Y.). Film images were thendigitized using a Portland scanner and figures were prepared by usingAdobe Photoshop (version 5.5): only brightness and contrast weremodified to create panels of comparable density in the finalillustrations.

D. Quantification and Statistical Analyses

To compare the effects of integrin antagonists on Aβ1-42internalization, quantification of Aβ1-42-ir elements was conducted forstained explants from four separate experiments that each included thefollowing treatments: Aβ1-42 only (n=8 explants), Aβ1-42 plus theinactive peptide GRADSP (n=9), Aβ1-42 plus the integrin antagonistpeptide GRGDSP (SEQ. ID. No.3) (n=19), or Aβ1-42 plus echistatin (n=12).Images centered on CA1b stratum pyramidale were obtained at 40×objective magnification using a Sony DKC-5000 digital camera attached toa Zeiss Universal microscope. The full digital images were then analyzedusing the densitometric capabilities of the National Institutes ofHealth Image 1.60 software on a G4 Macintosh computer. The “densityslice” option was used to threshold CA1 areas so that only immunolabeled“objects” were selected. The areas and mean density of selectedparticles for each image were then quantified using the “analyzeparticles” option. All images from the different treatment groups to becompared were digitized using the same acquisition parameters and thesame threshold density setting. Group mean values were obtained andstatistical significance was evaluated using the 2-tail Student's pairedt test. Significance was set at p<0.05.

II. Results

1. Uptake of Aβ1-42 is Enhanced by Integrin Antagonists

Cultured hippocampal slices were incubated in parallel for six daysunder one of the following treatment conditions: (1) vehicle (control);(2) Aβ1-42; (3) GRGDSP (SEQ. ID. No.3); and (4) Aβ1-42 plus GRGDSP (SEQ.ID. No.3), as described in Methods, and were then processed for theimmunohistochemical localization of incorporated human Aβ1-42. No Aβimmunostaining was detected in vehicle-treated control slices.Intraneuronal Aβ1-42 immunoreactivity (Aβ-ir) was detected in 42% [19/45] of the slices incubated with Aβ1-42 alone (Table 1). Staining wasrestricted almost exclusively to field CA1, where it was located inneuronal perikarya and proximal dendrites. As shown in FIG. 1, labelingwas found in discrete ‘medio-lateral’ segments of stratum pyramidale(see field bracketed by arrows in FIG. 1A). The restricted distributionof uptake is in agreement with previous reports (Bahr, B., et al., JComp Neurol 397:139-147 (1998)). Serial sections established the furtherpoint that labeling was densest in the superficial third of theAβ-treated slices (not shown). Examination at higher magnificationshowed that the antisera densely labeled a relatively homogenouspopulation of punctate bodies that were smaller than normal pyramidalcell somata (see FIG. 1B) and more lightly labeled a few processes inCA1 stratum radiatum. Based on the size and somatic location of theAβ-ir puncta, it appeared that immunostaining was concentrated inlysosomes. TABLE 1 Antagonist Effects on Internalization of Aβ₁₋₄₂ Aβ +Aβ + Aβ + Aβ + Control Aβ RGD RGD RAD Ech RGD number of 31 45 14 61 1621 16 slices tested Aβ positive 0 19 0 54 9 18 0 cases

The GRGDSP (SEQ. ID. No.3) peptide by itself did not induce Aβ-ir butdid markedly increase sequestration of the exogenous peptide. More than88% of the slices treated with Aβ1-42 plus 2 mM GRGDSP (SEQ. ID. No.3)[i.e., 54/61] had significant intraneuronal immunostaining (Table 1), apercentage that was significantly greater (p<0.0001, Fisher's test) thanthat found in explants treated with the Aβ peptide only. In furthercontrast to slices treated with Aβ-alone, labeling was detectablethroughout the depth of the slice and was both more dense and morebroadly distributed (FIG. 2A), in some instances extending into fieldsCA2 and CA3 (FIG. 3C). FIG. 2 further illustrates Aβ sequestrationpatterns in slices treated with Aβ1-42 plus GRGDSP (SEQ. ID. No.3).Labeling was uniformly dense throughout the pyramidal cell layer of CA1(FIG. 2A) but was not found in interneurons scattered throughout theapical and basal dendritic fields. Internalized Aβ-ir in CA1 pyramidalneurons formed discrete packets in the perinuclear cytoplasm (FIG.2B-D); these elements were noticeably larger than the punctatestructures stained in slices treated with Aβ alone. FIG. 2C illustratesthis point. The arrow points to a dense accumulation of Aβ1-ir thatstretches across the shaft of an apical dendrite; similar sized depositscan be seen in neighboring neurons. Immunopositive structures of thismagnitude were never found in the slices treated with Aβ alone.

FIG. 2C also illustrates the point that in slices treated with Aβ1-42plus GRGDSP (SEQ. ID. No.3), punctate Aβ-ir was accompanied by lighter,diffuse cytoplasmic immunostaining. As shown, Aβ-ir extended throughoutthe cell body and well into both apical and basal dendritic trees. Thebalance of punctate vs diffuse staining varied between hippocampalsubfields. FIG. 2D shows the border between fields CA1 and CA2 and, ascan be seen, discrete immunopositive structures were more prevalent inthe former region. In some cases, incorporated Aβ1-42 was found withinneurons that exhibited dendritic and axonal swellings (arrows in FIG.3E). FIG. 3F shows the dentate gyrus from a GRGDSP (SEQ. ID. No.3) plusAβ1-42-treated slice; Aβ1-ir was observed mainly in neurons scatteredthroughout the central and subgranular hilus although a few labeledcells were observed in stratum granulosum as well.

To further test the conclusion that GRGDSP (SEQ. ID. No.3) effects aredue to integrin antagonism, the effects of another small peptide,GRADSP, and of the disintegrin echistatin were examined. The GRADSPpeptide, which is a very weak antagonist of integrin binding tofibronectin and vitronectin and is typically used as an inactive controlpeptide (Pierschbacher, M., and Ruoslahti, E., Nature 304:30-33 (1984);Ruoslahti, E., Ann Rev Cell Dev Biol 12:697-715 (1996); Bahr, B. A., etal., J Neurosci 17:1320-1329 (1997)), did not measurably affectsequestration of AβB1-42 (FIG. 3A; Table 1). Disintegrins are small(4-10 kDa) RGD-containing, cysteine-rich peptides from snake venom thatbind integrins with high affinity (Kd˜1 nM-0.1 μM). They blockintegrin-mediated events in a wide variety of circumstances and are muchmore potent in this regard than GRGDSP (SEQ. ID. No.3) peptide (Huang,T. F., Cell Mol Life Sci 54:527-540 (1998) for a review). Previous workhas shown that injection of 5 μM echistatin with a microsplitzerselectively disrupts LTP stabilization in field CA1 of hippocampus (Chunet al., 2001). As shown in FIG. 3B, incubation with echistatin at 2 μMsignificantly enhanced Aβ internalization and, like GRGDSP (SEQ. ID.No.3) (FIG. 3C), expanded the zone of incorporation to include fieldCA3; Aβ1-42-ir was observed in 86% of echistatin plus Aβ1-42 treatedslices ( 18/21) (Table 1). At higher magnification one can see thedistribution of intraneuronal Aβ1-42-ir in echistatin treated slices(FIG. 3D) is similar to that induced by GRGDSP (SEQ. ID. No.3) (FIG.2D).

Quantitative analyses of levels of Aβ-ir in cultured slices verified theabove impressions of drug effects. As described in the Methods section,the “density slice” function of the NIH Image program was used tocalculate the numbers and overall area of Aβ-ir elements, at or above aspecific staining density, within a fixed-sized field of CA1 stratumpyramidale. The same density threshold was used for all slices from thedifferent treatment groups. FIGS. 4A-D show representative images fromslices treated with Aβ alone, Aβ plus GRADSP, Aβ plus echistatin, and Aβplus GRGDSP (SEQ. ID. No.3), respectively; in each case elements countedas being immunolabeled at or above the threshold density are highlightedin black. Results of quantitative analyses are shown in FIG. 4, graphs Ethrough G, which show the average area of individual Aβ1-ir particles(E), the total area encompassed by Aβ-ir elements (F) and the number ofAβ-ir elements within the sample field (G), respectively. Incubation ofhippocampal cultures with the integrin antagonists echistatin and GRGDSP(SEQ. ID. No.3) significantly enlarged the size of Aβ1-ir elements[+95±13% and +101±13% (mean ±sem); p<0.05 and p<0.01, respectively;2-tail t-test] (FIG. 4E) and increased the total area encompassed byimmunoreactive elements [233±24%, p<0.05 and 324±16%, p<0.01; 2-tailt-test, respectively] (FIG. 4F). Moreover, as shown in FIG. 4G, bothantagonists increased the numbers of Aβ-ir particles although thiseffect was statistically significant for GRGDSP (SEQ. ID. No.3) only.These results indicate that disruption of integrin mediated celladhesion not only increases intraneuronal levels of Aβ but also recruitsmore neurons into Aβ uptake processes. Incubation with the controlpeptide GRADSP did not cause significant changes in either the areas ornumbers of Aβ-ir elements (FIG. 4).

2. Enhanced Uptake of Aβ Increases Cathepsin D and Activates Microglia

Increases in cathepsin D are reported for AD brains (Cataldo, A. M., etal., Neuron 14:671-680 (1995); Callahan, L. M., et al., J NeuropatholExp Neurol 58:275-87 (1999); Ginsberg, S. D., et al., Ann Neurol48:77-87 (2000)) and were used to assess whether enhanced Aβ1-42sequestration produced by integrin antagonists generates age-relatedpathology. Six day treatments with Aβ1-42 by itself did not causesubstantial increases in cathepsin D-ir in any segment or collection ofcells within cultured slices (n=5; FIG. 5A). However, in the presence of2 mM GRGDSP (SEQ. ID. No.3), Aβ1-42 generated intense intraneuronalcathepsin D-ir (arrows in FIG. 5B). Elevated immunostaining waslocalized to puncta that had the distribution and approximate sizeexpected for lysosomes (FIG. 5C, inset). Labeling was more prominent in,but not restricted to, field CA1. Treatment with 2 mM GRGDSP (SEQ. ID.No.3) alone did not increase cathepsin D-ir.

In addition to the dense neuronal immunostaining, elevated cathepsinD-ir was also localized in patches within small irregularly distributedcells that had short processes and small cell bodies (arrowheads in FIG.5C). Based on their size, distribution and morphology, these cathepsinD-ir cells appeared to be microglia. Immunopositive elements of thistype were not observed in slices treated with Aβ1-42 or GRGDSP (SEQ. ID.No.3) alone.

Alzheimer disease is also associated with a brain inflammatory response[see (Akiyama, H., et al., Neurobiol Aging 21:383-421 (2000)) for arecent review], one component of which involves microglia (Cras, P., etal., Brain Res 558:312-314 (1991)). To test if uptake of Aβ inducesinflammatory activity, and specifically activates microglial cells,double staining was carried out using anti-Aβ1-42 sera and monoclonalantibody ED-1, a marker for lysosomal membranes within reactivemicroglia (Kato, H., et al., Brain Res 694:85-93 (1995); Woods, A. G.,et al., Neurosci 91:1277-1289 (1999)); this yielded dark grey and blackreaction products for Aβ-ir and ED-1ir, respectively (FIG. 6). Aβ1-42and GRGDSP (SEQ. ID. No.3) by themselves did not cause a noticeableincrease in the number of ED-1 positive cells but applied togetherproduced a pronounced microglial response (FIG. 6A). ED-1-ir microgliawere observed mainly in fields CA1 (FIG. 6B) and CA3c (FIG. 6C); thesecells had “reactive” features including a rounded and enlarged cell bodyand shortened processes. The Aβ1-42- and ED-1-immunoreactivities werenot colocalized within individual cells indicating that the microgliadid no internalize Aβ at detectable levels. Importantly, however, ED-1positive microglia were co-distributed with Aβ-ir neurons. That is,large numbers of labeled microglia were found intermingled withpopulations of Aβ immunostained neurons. These co-distributed cells weremost frequently found in subregions of field CA1 but fields ofoverlapping labeled cells were occasionally distributed within in CA2,CA3 and subiculum as well. Areas lacking Aβ-ir neurons were nearlydevoid of ED-1 positive microglia, as shown on the left side of FIG. 6D.

3. Uptake and Effects of Aβ-42 are Blocked by an NMDA ReceptorAntagonist

Activation of the NMDA-class glutamate receptors allows calcium influxand facilitates internalization of membrane protein (Carroll, R. C.,Proc Natl Acad Sci USA 96:14112-14117 (1999); Beattie, E. C., et al.,Nat Neurosci 3:1291-300 (2000); Ehlers, M. D., Neuron 28:511-25 (2000)).A selective antagonist of NMDA receptors, AP5, was used to test if NMDAreceptors are involved in GRGDSP-enhanced (SEQ. ID. No.3) Aβ1-42 uptake.FIG. 7 shows Aβ1-42 immunostaining in sections from slices that had beenincubated in parallel with Aβ1-42 plus GRGDSP (SEQ. ID. No.3) with (FIG.7A′) or without (FIG. 7A) AP5. As shown, the NMDA receptor antagonistcompletely blocked Aβ1-42 uptake. Comparable results were obtained in 16experiments, the total of this type conducted.

FIGS. 7B′ and 7B compare immunostaining for cathepsin D in slicesincubated with Aβ1-42 and GRGDSP (SEQ. ID. No.3) in the presence orabsence of AP5, respectively. As shown, intraneuronal cathepsin D-ir wasalmost totally suppressed in the slice exposed to AP5 (FIG. 7B′). Notealso that cathepsin D immunostaining of presumed microglia is alsoabsent from the experimental slice, as expected if this effect wassecondary to neuronal uptake and consequent neuronal pathology.Similarly, ED-1-ir microglia were also greatly reduced in AP-5co-treated slices (FIG. 7C′). Thus, each of the AD-like changes thatotherwise accompany GRGDSP-enhancement (SEQ. ID. No.3) of Aβ1-42 uptakewere blocked by the NMDA receptor antagonist AP5.

III. Discussion

Uptake of Aβ is Enhanced by RGD-Binging Integrin Antagonists

The principal integrin antagonist used herein (i.e., GRGDSP (SEQ. ID.No.3)) was reported to block the binding and uptake of Aβ in CHO cellsin culture (Matter, M. L., et al., J Cell Biol 141:1019-1030 (1998))but, as described here, markedly enhances uptake in cultured hippocampalslices. The majority of slices under control conditions did notaccumulate appreciable concentrations of Aβ1-42 during six-dayincubations. Moreover, when substantial uptake did occur, it wasrestricted both with regard to depth in the slice and to hippocampalsubdivision (i.e., CA1 stratum pyramidale), which is in agreement withthe previous studies (Bahr, B., et al., J Comp Neurol 397:139-147(1998); Harris-White, M. E., et al., J Neurosci 18:10366-10374 (1998)).Thus, relatively mature neurons surrounded by diverse glia andpossessing large synaptic populations appear to effectively resistuptake and/or accumulation of Aβ1-42. Treatment with integrinantagonists led to substantial intraneuronal buildup of Aβ1-42 andexpanded the slice depth and anatomical range over which uptakeoccurred.

Enhanced Intraneuronal Aβ Levels Upregulate Cathepsin D and ActivateMicroglia

Enhanced accumulation of Aβ1-42 was accompanied by a marked increase inintraneuronal cathepsin D and activation of microglia. Previousbiochemical studies showed that Aβ1-42 causes a modest increase incathepsin D concentrations in cultured slices (Hoffman, K. B., et al.,Neurosci Lett 250:75-78 (1998)); the present results indicate that thiseffect is markedly enhanced when the uptake and/or accumulation ofamyloid is increased. Experimentally induced lysosomal dysfunctioncauses a rapid increase in the concentration and activity of cathepsin Din cultured slices (Bednarski, E., and Lynch, G., Neuroreport9:2089-2094 (1998); Bi, X., et al., J Neurochem 74:1469-1477 (2000)) andstudies with dissociated neurons have shown that Aβ1-42 triggers therelease of the protease into the cytoplasm (Yang, A. J., et al., JNeurosci Res 52:691-698 (1998)). In addition to showing thatinternalized Aβ causes significant intracellular disturbances, theincreases of cathepsin D observed herein suggest links between Aβsequestration and AD pathologies. That is, increases in Cathepsin Doccur in AD-vulnerable neurons in advance of overt pathology (Cataldo,A. M., et al., Neuron 14:671-680 (1995)) and correlate, on acell-by-cell basis, with the presence of neurofibrillary tangles anddecreases in synaptophysin in field CA1 of AD brains (Callahan, L. M.,et al., J Neuropathol Exp Neurol 58:275-87 (1999); Ginsberg, S. D., etal., Ann Neurol 48:77-87 (2000)).

The microglial reaction observed in the present study may constitute asecond link between effects of Aβ sequestration and AD. Inflammation,including microglial activation, is now recognized as an importantcomponent of AD-related pathology [see (Akiyama, H., et al., NeurobiolAging 21:383-421 (2000)) for review]. Amyloid plaques are typicallysurrounded by reactive microglia (Perlmutter, L. S., et al., NeuroscLett 119:32-36 (1990); Cras, P., et al., Brain Res 558:312-314 (1991))as are Aβ deposits in brains of 12 month old AβPP(V717F) transgenic mice(Murphy, G. M. J., et al., Am J Pathol 157:895-904 (2000)). In theinstant invention, microglia were similarly spatially associated withAβ, but in this instance, as described, Aβ1-42 was accumulatedintraneuronally. This suggests that internalized Aβ triggers pathogenicresponses in neurons, resulting in the release of signals that activatemicroglia. Finally, the presence of both microglial activation andcathepsin D induction in brain tissue with heightened Aβ uptake, supportthe hypothesis that the low levels of uptake occurring with exposure toAβ peptide alone may be responsible for the relatively modest effects ofexogenous Aβ on complex brain systems (Games, D., et al., NeurobiolAging 13:569-576 (1992); Podlisny, M. B., et al., Am J Pathol 142:17-24(1993); Bahr, B., et al., Comp Neurol 397:139-147 (1998); Harris-White,M. E., et al., J Neurosci 18:10366-10374 (1998)).

It has generally been accepted that insoluble aggregated fibrilsconsisting of Aβ1-42 are the neurotoxic components in AD pathogenesis.However, in vitro experiments revealed that extracellular Aβconcentrations, even in familial AD (Suzuki, N., et al., Science264:1336-1340 (1994); Kuo, Y. M., et al., J Biol Chem 271:4077-4081(1996); Scheuner, D., et al, Nat Med 2:864-870 (1996)), are far belowwhat is necessary for fibrillar aggregation (Harper, J. D., andLansbury, P. T. J., Annu Rev Biochem 66:385-407 (1997)). On the otherhand, intracellular Aβ concentrations may reach amyloidogenic levels(Yang, A. J., et al., J Neurosci Res 52:691-698 (1998); Gouras, G. K.,et al, Am J Pathol 156:15-20 (2000); Walsh, D. M., et al., Biochemistry39:10831-10839 (2000)) through overproduction, enhanced internalization,or decreased degradation. High concentrations of intracellular Aβ couldaggregate, cause neurodegeneration, and “seed” mature neuritic plaques.Intraneuronal initiation of Aβ toxicity is supported by recenttransgenic studies showing that the peptide can cause synapticdegeneration in the absence of extracellular plaque formation (Hsia, A.Y., et al., Proc Natl Acad Sci USA 96:3228-3233 (1999); Mucke, L., al.,J Neurosci 20:4050-8 (2000)). Disassociation of extracellular Aβdeposition and functional impairment was also observed in recent vaccinestudies: while immunization with Aβ significantly blocked learning andmemory deficits in transgenic mice, it only modestly decreasedextracellular Aβ deposits (Morgan, D., et al., Nature 408:982-85(2000)).

NMDA Receptors are Involved in Integrin Antagonist-Induced aβ Uptake andCorrelates

The present experiments also demonstrate that NMDA receptors have apotent effect on Aβ uptake. NMDA receptors admit calcium into neurons(Burnashev, N., et al., Science 257:1415-1419 (1992)) and are coupled tothe intracellular actin network (Dunah, A. W., et al., Brain Res MolBrain Res 79:77-87 (2000)). Calcium is critical to endocytosis (Seiler,C., and Nicolson, T., J Neurobiol 41:424-434 (1999)) while the actinnetwork appears to be a central component of endocytosis across celltypes (Gottlieb, T. A., et al., J Cell Biol 120:695-710 (1993)).Moreover, recent studies have shown that activation of NMDA receptors bysynaptically released glutamate promotes clathrin-coated pit endocytosisin cultured neurons (Carroll, R. C., Proc Natl Acad Sci USA96:14112-14117 (1999); Beattie, E. C., et al., Nat Neurosci 3:1291-300(2000); Ehlers, M. D., Neuron 28:511-25 (2000)).

The actions of the NMDA receptor antagonist on the uptake of Aβ wereparalleled by its effects on Aβ-induced increases in cathepsin D andmicroglial activation. This constitutes strong evidence that both ofthese pathogenic effects were secondary to intraneuronal accumulation ofAβ. The results also raise the possibility that a gradual loss ofintegrin-mediated adhesion with age results in enhanced contributions ofthe NMDA receptor to Aβ internalization and, hence, in a slow increasein amyloid-triggered pathogenesis. There is evidence that integrinmediated adhesion changes with age in peripheral cells (Le Varlet et al,1998; Labat-Robert, 1998) but similar analyses have not been conductedfor brain; comparable age-effects at brain synapses could result inenhancement of NMDA receptor driven effects of the type seen in integrinantagonist-treated slices herein.

The above results provide, among other things, the following. 1)Sequestration of and/or accumulation of and/or uptake of Aβ can beinduced in culture slices in a medium which modulates integrins and/orintegrin receptors. 2) Incubating cultured hippocampal slices with anagent which modulates integrins and/or integrin receptors for 4 daysresulted in the sequestration of and/or accumulation of and/or uptake ofAβ that was far more significant than in hippocampal slices not treatedwith an agent which modulates integrins and/or integrin receptors. 3)Immunocytochemical analysis revealed that the hippocampal slices thatwere treated with an agent which modulates integrins and/or integrinreceptors had enhanced levels of cathepsin D when compared tohippocampal slices not treated with an agent which modulates integrinsand/or integrin receptors. 4) Activation of microglia was significantlygreater in hippocampal slices that were treated with an agent whichmodulates integrins and/or integrin receptors when compared tohippocampal slices not treated with an agent which modulates integrinsand/or integrin receptors. 5) Contacting the brain cells withantagonists to glutamate receptors, such as the NMDA-type glutamatereceptor, significantly attenuated any and/or all of the effectstriggered by contacting the slices with agent(s) which can modulateintegrins and/or integrin receptors.

Thus, the present invention provides an assay system wherein the levelsof sequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation have beensignificantly induced in brain cells. Moreover, the present inventionprovides clear evidence, for the first time, for the relationshipbetween integrins and/or integrin receptors, and the sequestration ofand/or accumulation of and/or uptake of Aβ, one of the major pathologiesin neurodegenerative diseases such as Alzheimer's disease. The locationof the sequestration of and/or accumulation of and/or uptake of Aβ,and/or lysosomal dysfunction, and/or microglia activation corresponds tothat in tissues from Alzheimer's disease patient.

Among other things, the present invention provides that thesequestration of and/or accumulation of and/or uptake of Aβ, and/orlysosomal dysfunction, and/or microglia activation can be induced inbrain cells by contacting the brain cells with a medium that modulatesintegrins and/or integrin receptors. Moreover, the present resultsdemonstrated that contacting such tissue with antagonists to glutamatereceptors in brain, such as the NMDA receptor, can significantlyattenuate the tissue's susceptibility to Aβ and its associatedpathologies. These results significantly extend the range ofneurodegenerative disease features that can be induced in brain cellculture.

In vitro and in vivo tests have demonstrated that amyloid plays anintegral role in the pathogenesis of neurodegenerative disease. Thus,not wishing to be bound by a theory, the uptake and/or internalizationof amyloid could be a key factor in the pathologies associated with suchdiseases, and therefore the inhibition of amyloid internalization and/oruptake by the application of an antagonist to glutamate receptors may bea viable therapeutic option for diseases and disorders comprisingpathologies related to amyloid. These results significantly extend therange of neurodegenerative disease features that can be induced and/orinhibited in brain cells.

EXAMPLE 2

An embodiment of the invention drawn to a pharmaceutical composition andthe use of that composition to treat neurodegenerative diseases such asAlzheimer's disease. The composition alleviates the symptoms ofcharacteristics associated with Alzheimer's disease such asintracellular uptake of amyloid protein, amyloid accumulation or plaqueformation,

A patient in need of intervention for Alzheimer's disease is selectedbased on currently used diagnostic guidelines and evaluation criteriasuch as those detailed: by the National Institute of Neurological andCommunicative Disorders and Stroke-Alzheimer's Disease and relatedDisorders Association (NINCDS-ADRDA), the Alzheimer's Disease sectionfound in the Diagnostic and Statistical Manual of Mental Disorders4^(th) edition (DSM-IV), by the National Institute of NeurologicalDisorders and Stroke and the Association pour la REcherche etl'Enseignement en Neurosciences (NINDS-AIREN), and/or by the CaliforniaAlzheimer's Diseases Diagnostic and Treatment Centers (CAD-DTC).

After evaluation the patient is treated with a pharmaceuticalcomposition comprising an appropriate amount of an NMDA antagonist suchas magnesium, ketamine, dextromethorphan, amantadine, dexanabinol, AP3,AP5, AP6, AP7, 4C3HPG, 4CPG, CGS 19755, chlorophenylglutamic acid, CPP,MK-801, PCP, ibogaine, noribogaine, ienprodil, flupirtine, selfotel,D-CPP-ene, procyclidine, trihexyphenidyl, CP-101606, CP-98113,GVI150526, AR-R15896AR, NPS 1506, NPC 12626, LY274614, LY 2835959, SDZ220-040, SDZ 220-040, SDZ 220-581, SDZ 221-653, and similar compounds,based on the body weight of the patient and with an appropriate carrieror excipient.

Those of skill in the art would be able to obtain general and sourceinformation for the above compounds. Specific information several ofthese antagonists can be found in; Curr. Drug Targets 2:241-271 (2001),Rao et al, Brain Res. 911:96-100 (2001) (memantine), Mueller et al.,Ann. N.Y. Acad Sci. 890:450-457 (1999), Chazot Curr. Opin. Investig.Drugs 1:370-374 (2000) and Proescholdt et al., Brain Res. 904:245-251(2001) (ketamine). Specific commercial sources for AP5 and MK801 areSIgman and Research Biochemical Incorporation respectively.

Following a period of treatment the patient is reevaluated and treatmentis continued with increasing or decreasing amounts of the pharmaceuticalcomposition.

Evaluations are carried out using techniques known to those of skill inthe art and may include MRI (Selkoe, D. Nat. Biotechnol. 18:823-824,2000; Fox, N. et al., Nat. Med. 6:20-21, 2000), PET,Electroencephalogram analysis and use of certain cognitive tests(Clinical Demention Rating (DR), Morris, J. Neurology 43:2412-2414,1993).

The present invention provides novel materials, such as brain cells andmethods for producing the cells which can be used as a model ofneurodegenerative diseases, including Alzheimer's disease. Additionally,methods of treating neurodegenerative diseases are provided. Whilespecific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, applicants do not admit any particular reference is “priorart” to their invention.

1. A method for determining the effect of a substance on characteristics of neurodegenerative disease in brain cells, said method comprising: (A) exposing brain cells to a condition that modulates integrins or integrin receptors in said cells, (B) maintaining said cells for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells, (C) adding said substance before, during and/or after said exposing or maintaining; and (D) determining whether the presence of said substance has an effect on one or more of said characteristics.
 2. The method of claim 1 wherein said characteristics are selected from the group consisting of: (1) sequestration of Aβ, (2) accumulation of Aβ, (3) uptake of Aβ, (4) lysosomal dysfunction, (5) microglia activation, and (6) changes in cathepsin D content.
 3. The method of claim 2, wherein at least one of said characteristics increases.
 4. The method of claim 3, wherein said increase is at least about 10% compared to a control.
 5. The method of claim 2, wherein at least one of said characteristics decreases.
 6. The method of claim 5, wherein said decrease is at least about 10% compared to a control.
 7. The method of claim 1, wherein the brain cells are in the form of a brain slice.
 8. The method of claim 7, wherein the brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.
 9. The method of claim 1 wherein said brain cells are in vivo.
 10. The method of claim 1, wherein the brain cells are from a non-human transgenic animal.
 11. The method of claim 10, wherein said non-human transgenic animal comprises a human apolipoprotein E4 gene.
 12. The method of claim 10 wherein both alleles of an endogenous apolipoprotein E gene of the non-human transgenic animal are ablated.
 13. The method of claim 1, wherein said brain cells in step (A) are cultured in a medium that comprises an antagonist or modulator of an integrin.
 14. The method of claim 13 wherein said antagonist or modulator of integrin is a neutralizing or function blocking antibody for integrin subunits wherein said subunits are selected from the consisting of: alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, and alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 and beta8.
 15. The method of claim 13, wherein said antagonist or modulator of integrin comprises a compound selected from the group consisting of RGD, RGDS (SEQ. ID. No.1), GRGDS (SEQ. ID. No.2), GRGDTP (SEQ. ID. No.4) and GRGDSP (SEQ. ID. No.3), mimetics thereof, echistatin, trilavin, disintegrins and snake venom.
 16. The method of claim 2, wherein the amount of, sequestration of Aβ, accumulation of Aβ, uptake of Aβ, lysosomal dysfunction, levels of cathepsin D or microglia activation is determined visually.
 17. The method of claim 2, wherein the amount of; sequestration of Aβ, accumulation of Aβ, uptake of Aβ, lysosomal dysfunction, change of cathepsin D content or microglia activation is measured using a capture reagent.
 18. The method of claim 16, wherein the capture reagent is an antibody that binds to Aβ, lysosomes, Cathepsin D or a microglia element.
 19. The method of claim 1 wherein said cells are apolipoprotein E deficient brain cells or apolipoprotein E4 containing brain cells cultured in a medium which selectively increases sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation in the brain cells, wherein the brain cells comprise an increased amount of sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation compared to a control.
 20. A method of obtaining brain cells having characteristics of neurodegenerative disease comprising: (A) culturing brain cells, (B) exposing said brain cells to a condition that modulates integrins or integrin receptors in said cells, and (C) maintaining said cells or brain tissue for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells.
 21. The method of claim 20, wherein said characteristics are selected from the group consisting of: (1) sequestration of Aβ, (2) accumulation of Aβ, (3) uptake of Aβ, (4) lysosomal dysfunction, (5) microglia activation and (6) changes in cathepsin D content.
 22. The method of claim 21, wherein at least one of said characteristics increases.
 23. The method of claim 22, wherein said increase is at least about 10% compared to a control.
 24. The method of claim 21, wherein at least one of said characteristics decreases.
 25. The method of claim 24, wherein said decrease is at least about 10% compared to a control.
 26. The method of claim 20, wherein the brain cells are in the form of a brain slice.
 27. The method of claim 26, wherein the brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.
 28. The method of claim 20, wherein the brain cells are from a non-human transgenic animal.
 29. The method of claim 28, wherein said non-human transgenic animal comprises a human apolipoprotein E4 gene.
 30. The method of claim 28 wherein both alleles of an endogenous apolipoprotein E gene of the non-human transgenic animal are ablated.
 31. The method of claim 20, wherein said brain cells in step A are cultured in a medium that comprises an antagonist or modulator of an integrin.
 32. The method of claim 31 wherein said antagonist or modulator of integrin is a neutralizing or function blocking antibody for integrin subunits wherein said subunits are selected from the group consisting of: alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, and alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 and beta8.
 33. The method of claim 31, wherein said antagonist or modulator of integrin comprises a compound selected from the group consisting of RGD, RGDS (SEQ. ID. No.1), GRGDS (SEQ. ID. No.2), GRGDTP (SEQ ID NO.4) and GRGDSP (SEQ. ID. No.3), mimetics thereof, echistatin, triflavin, disintegrins and snake venom.
 34. The method of claim 20 wherein said cells are apolipoprotein E deficient brain cells or apolipoprotein E4 containing brain cells cultured in a medium which selectively increases sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation in the brain cells, wherein the brain cells comprise an increased amount of sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation compared to a control.
 35. Brain cells obtained by the method of claim
 20. 36. The method of claim 1 wherein the brain cells are contacted with a compound that modulates integrins or integrin receptors prior to contacting with the substance whose effect is being determined.
 37. The method of claim 1, wherein the brain cells are contacted simultaneously with the compound that modulates integrins and/or integrin receptors and the substance whose effect is being determined.
 38. An in vitro method for increasing at least one or more characteristics of neurodegenerative disease in brain cells, wherein said characteristics are selected from the group consisting sequestration of Aβ, accumulation of Aβ, uptake of Aβ, lysosomal dysfunction, changes in cathepsin D content and microglia activation, said in vitro method comprising: (A) exposing brain cells in culture to a condition that modulates integrins or integrin receptors in said cells wherein said modulation results in increase in characteristics of neurodegenerative disease in said cells, and (B) maintaining said cells in culture for a time sufficient to increase one or more characteristics of a neurodegenerative disease in said cells.
 39. The method of claim 38, wherein at least one of said characteristics increases.
 40. The method of claim 39, wherein said increase is at least about 10% compared to a control.
 41. The method of claim 38, wherein at least one of said characteristics decreases while other characteristics increase.
 42. The method of claim 41, wherein said decrease is at least about 10% compared to a control.
 43. The method of claim 38, wherein the brain cells are in the form of a brain slice.
 44. The method of claim 43, wherein the brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.
 45. The method of claim 38, wherein the brain cells are from a non-human transgenic animal.
 46. The method of claim 45, wherein said non-human transgenic animal comprises a human apolipoprotein E4 gene.
 47. The method of claim 45 wherein both alleles of an endogenous apolipoprotein E gene of the non-human transgenic animal are ablated.
 48. The method of claim 38, wherein said brain cells in step (A) are cultured in a medium that comprises an antagonist of an integrin.
 49. The method of claim 48 wherein said antagonist or modulator of integrin is a neutralizing or function blocking antibody for integrin subunits wherein said subunits are selected from the group consisting of: alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, and alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 and beta8.
 50. The method of claim 48 wherein said antagonist or modulator of integrin comprises a compound selected from the group consisting of RGD, RGDS (SEQ. ID. No.1), GRGDS (SEQ. ID. No.2), GRGDTP (SEQ. ID. No.4) and GRGDSP (SEQ. ID. No.3), mimetics thereof, echistatin, triflavins, disintegrins and snake venom.
 51. The method of claim 38 wherein said cells are apolipoprotein E deficient brain cells or apolipoprotein E4 containing brain cells cultured in a medium which selectively increases sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation in the brain cells, wherein the brain cells comprise an increased amount of sequestration of and/or accumulation of and/or uptake of Aβ, and/or lysosomal dysfunction, and/or microglia activation compared to a control.
 52. The brain cells produced by the method of claim
 38. 53. Brain cells in vitro, wherein the brain cells have been treated with a compound that that modulates integrins and/or integrin receptors, thereby producing characteristics of a brain afflicted with a neurodegenerative disease or an aged brain.
 54. The brain cells of claim 53, wherein said characteristics are selected from the group consisting of sequestration of Aβ, accumulation of Aβ, uptake of Aβ, lysosomal dysfunction, changes in cathepsin D content and microglia activation.
 55. A method for alleviating the symptoms of disease states having at least one of the following characteristics selected from the group consisting of intracellular uptake of amyloid protein, amyloid accumulation and plaque formation, said method comprising: (A) administering to a patient in need thereof a composition comprising an effective amount of an NMDA receptor antagonist, and (B) determining the effectiveness of treatment with said composition, (C) increasing or decreasing the composition based on the determinative testing, and (D) alleviating symptoms of the disease.
 56. A pharmaceutical composition comprising a compound capable of sufficiently inhibiting the activity of the NMDA receptor in an amount effective to alleviate one or more symptoms of disease states associated with at least one characteristic selected from the group consisting of abnormal accumulation, abnormal molecular organization of amyloid protein and amyloid plaques and said composition also includes a suitable carrier or pharmaceutical excipient.
 57. The pharmaceutical composition of claim 56 wherein said compound is selected from a group consisting of magnesium, ketamine, dextromethorphan, amantadine, dexanabinol, AP3, AP5, AP6, AP7, 4C3HPG, 4CPG, CGS 19755, chlorophenylglutamic acid, CPP, MK-801, PCP, ibogaine, noribogaine, ifenprodil, flupirtine, selfotel, D-CPP-ene, procyclidine, trihexyphenidyl, CP-101606, CP-98113, GVI150526, AR-R15896AR, NPS 1506, NPC 12626, LY274614, LY 2835959, SDZ 220-040, SDZ 220-040, SDZ 220-581, SDZ 221-653 and memantine.
 58. A method for inhibiting the intracellular accumulation of amyloid comprising: (A) contacting brain cells with a glutamate receptor antagonist and (B) determining whether the intracellular accumulation of amyloid is inhibited.
 59. A method for determining the effect of a substance on inhibition of characteristics of neurodegenerative disease in brain cells, said method comprising: (A) exposing brain cells to a condition that modulates integrins or integrin receptors in said cells, (B) maintaining said cells for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells, (C) adding said substance before, during and/or after said exposing or maintaining; and (D) determining whether the presence of said substance inhibits one or more of said characteristics.
 60. The method of claim 59 wherein said characteristics are selected from the group consisting of: (1) sequestration of Aβ, (2) accumulation of Aβ, (3) uptake of Aβ, (4) lysosomal dysfunction, (5) microglia activation and (6) changes in cathepsin D content.
 61. The method of claim 60, wherein at least one of said characteristics decreases.
 62. The method of claim 61, wherein said decrease is at least about 10% compared to a control.
 63. The method of claim 60, wherein the brain cells are in the form of a brain slice.
 64. The method of claim 63, wherein the brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.
 65. The method of claim 59 wherein said brain cells are in vivo.
 66. The method of claim 59, wherein the brain cells are from a non-human transgenic animal.
 67. The method of claim 66, wherein said non-human transgenic animal comprises a human apolipoprotein E4 gene.
 68. The method of claim 67 wherein both alleles of an endogenous apolipoprotein E gene of the non-human transgenic animal are ablated.
 69. The method of claim 59, wherein said brain cells in step A are cultured in a medium that comprises an antagonist or modulator of an integrin.
 70. The method of claim 69 wherein said antagonist or modulator of integrin is a neutralizing or function blocking antibody for integrin subunits wherein said subunits are selected from the group consisting of: alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, and alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 and beta8
 71. The method of claim 69, wherein said antagonist or modulator of integrin comprises a compound selected from the group consisting of RGD, RGDS (SEQ. ID. No.1), GRGDS (SEQ. ID. No.2), GRRDT (SEQ. ID. No.4) and GRGDSP (SEQ. ID. No.3), mimetics thereof, echistatin, triflavin, disintegrins and snake venom.
 72. The method of claim 60, wherein the amount of; sequestration of Aβ, accumulation of Aβ, uptake of Aβ, lysosomal dysfunction, changes in cathepsin D content or microglia activation is determined visually.
 73. The method of claim 60, wherein the amount of, sequestration of Aβ, accumulation of Aβ, uptake of Aβ, lysosomal dysfunction or microglia activation is measured using a capture reagent.
 74. The method of claim 73, wherein the capture reagent is an antibody that binds to Aβ, lysosomes, Cathepsin D or a microglia element.
 75. The method of claim 59 wherein said cells are apolipoprotein E deficient brain cells or apolipoprotein E4 containing brain cells.
 76. The method of claim 55 wherein said determining is done using brain imaging techniques.
 77. The method of claim 76 wherein said brain imaging techniques are MRI or PET.
 78. The method of claim 55 wherein said determining is done using EEG or cognitive tests. 